Oxadiazolo[3,2-a]pyrimidines and thiadiazolo[3,2-a]pyrimidines

ABSTRACT

The present invention relates to compounds of formula P-1 and related compounds and compositions useful for inhibiting and/or reducing platelet deposition, adhesion and/or aggregation. The definitions of A, Y, R 1 , R 2 , R a , R a′ , R b , R b′ , R c , R c′ , R d , R d′ , R e , and R e′  are provided herein. The present invention further relates to methods for the treatment or prophylaxis of thrombotic disorders, including stroke, myocardial infarction, unstable angina, peripheral vascular disease, abrupt closure following angioplasty or stent placement and thrombosis as a result of vascular surgery.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a United States Application under 35 U.S.C. 371claiming benefit of PCT Application No. PCT/US2011/044267, filed on Jul.15, 2011, which claims priority from U.S. Provisional Application No.61/365,152, filed on Jul. 16, 2010, the contents of which are herebyincorporated by reference in their entirety.

The present invention was made with funding from National Institutes ofHealth Grant No. HL019278 and NIH/NHLBI R03MH83257-1. The United StatesGovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to methods for screening compounds andcompositions useful for inhibiting or reducing platelet deposition,adhesion and/or aggregation. The present invention further relates tomethods of treatment or prophylaxis of thrombotic disorders, includingstroke, myocardial infarction, unstable angina, abrupt closure followingangioplasty or stent placement, thrombosis induced by peripheralvascular surgery, peripheral vascular disease or thrombotic disordersresulting from atrial fibrillation or inflammation.

BACKGROUND

Platelet accumulation at sites of vascular injury is a dynamic processthat mediates formation of both the primary hemostatic plug andpathologic thrombus formation. The mechanisms by which platelet surfaceproteins direct platelet recruitment to thrombi under flow conditionshave been studied in detail. In addition to directing initial plateletadhesion, cell-surface receptor interactions activate intracellularsignaling. Intracellular signaling stimulates the release ofthrombogenic substances from platelet granules. Signaling also mediatesactivation of the platelet integrin γIIbβ3 that facilitates firmadhesion of the platelets at the sites of injury.

Arterial thrombosis mediates tissue infarction in coronary arterydisease, cerebrovascular disease, and peripheral vascular disease, and,thus, is the single most common cause of morbidity and mortality in theUnited States. Platelets are key mediators of arterial thrombosis. Thus,the identification of compounds that inhibit platelet function is ofgreat importance to medicine.

Platelets form the body's primary means of hemostasis and, as such, havedeveloped an elaborate mechanism of surveying the vasculature fordefects in endothelial integrity. This mechanism involves the ability torespond to subendothelial matrices, shear forces, neighboring platelets,the adrenal axis, as well as soluble proteinacious, nucleotide, andlipid signals. Despite this plethora of physiologic activators, theplatelet has only a small repertoire of major functional outputs. Uponactivation, platelets change shape, aggregate, and secrete theirgranular contents. The process of platelet activation involves theexpression of activities not shared by functionally intact restingplatelets, including, for example, ATP release, serotonin release,lysosomal release, alpha granule release, dense granule release, andcell surface expression of markers of activated platelets [including,but not limited to P-selectin and activated αIIbβ3 (GPIIb/IIIa)receptor]. In addition, platelet activation results in the aggregationof platelets with each other and attachment to non-platelet surroundingcells. The granular contents of platelets supply additional adhesionmolecules, growth factors, coagulation enzymes and other specializedmolecules instrumental in the process of thrombus formation and theinitiation of the healing process.

In addition to coronary artery disease/myocardial infarction,cerebrovascular disease and peripheral vascular disease, diseases anddisorders associated with inappropriate platelet activity and arterialthrombosis also include, for example, stable and unstable angina,transient ischemic attacks, placental insufficiency, unwanted thrombosessubsequent to surgical procedures (e.g., aortocoronary bypass surgery,angioplasty and stent placement, and heart valve replacement), orthromboses subsequent to atrial fibrillation. Inhibitors of plateletactivity can provide therapeutic and preventive benefits for each ofthese diseases or disorders. It is also possible that inappropriateplatelet activation plays a role in venous thrombosis, such thatplatelet inhibitors can be useful for the treatment or prophylaxis ofdisorders associated with such thromboses.

A connection is emerging between platelet activation and inflammation,particularly allergic inflammation (e.g., in asthma) and inflammation atthe sites of atherosclerotic damage. Therefore, compounds that inhibitplatelet activation can also be useful in the treatment or prophylaxisof disorders involving inflammation.

There are a number of agents presently available that target plateletfunction. For example, aspirin is a relatively weak platelet inhibitor.However, aspirin can cause life-threatening allergic reactions insensitive individuals.

Another platelet inhibiting agent is ticlopidine (Ticlid™, RochePharmaceuticals). Because it requires the production of activemetabolites to be effective, the effect of ticlopidine is delayed 24-48hours. The drug can also cause thrombotic thrombocytopenic purpura aswell as life threatening leukopenia, nausea, abdominal pain, dyspepsia,diarrhea and skin rash.

Clopidogrel (Plavix™, Bristol-Meyers Squibb/Sanofi Pharmaceuticals) isanother platelet inhibitor that requires the generation of activemetabolites for its therapeutic efficacy. Therefore, clopidogrel alsohas a delay of at least several hours for its effect. Clopidogrel canalso cause thrombotic thrombocytopenia purpura. The drug has also beenassociated with a number of side effects, including rash, edema,hypertension, hypercholesterolemia, nausea, abdominal pain, dyspepsia,diarrhea, urinary tract infections, liver enzyme elevations andarthralgia.

Recently, prasugrel was approved as a P2Y₁₂ inhibitor for use as aplatelet inhibitor, but similar to clopidogrel, major bleeding,including non-fatal as well as fatal bleeding was observed.

The platelet inhibitory agent abciximab (c7E3 Fab, Reopro®,manufacturer-Centocor B. V., distributor-Eli Lilly and Co.) is onlyavailable in a parenteral form. The drug can cause severethrombocytopenia. Its antiplatelet effects last for several days unlessplatelet transfusions are given and, therefore, may complicate surgerythat is sometimes required in the setting of life-threatening arterialocclusion (e.g., emergent cardiac surgery in the setting of a myocardialinfarction).

There is only limited clinical experience with the oral anti-αIIbβ3agents lamifiban, sibrafiban, orbofiban and xemilofiban, none of whichare approved for human use. Similarly, clinical experience is limitedwith the phosphodiesterase inhibitors cilostazol, trapidil and trifusal.There is more clinical experience with the phosphodiesterase inhibitordipyridamole, but its activity is relatively weak and so it is notfrequently used unless combined with aspirin.

There is a need in the art for additional platelet adhesion andaggregation inhibitory agents for the treatment and prophylaxis ofdiseases or disorders associated with abnormalities in platelet adhesionand aggregation.

It is known that integrin αIIbβ3 is a receptor on the surface of humanplatelets. As a heterodimeric complex composed of both αIIb and β3subunits, the dimer is responsible for binding adhesive plasma proteins,most notably fibrinogen and von Willebrand factor (vWF). The binding offibrinogen, vWF and other ligands by αIIbβ3 is mediated principallythough the peptide recognition sequence Arg-Gly-Asp (RGD) or thefibrinogen γ chain dodecapeptide HHLGGAKQAGDV. Conformational changes inαIIbβ3 are thought to occur upon the binding of ligand to the receptor,leading to the exposure of ligand-induced binding sites (LIBS) asdetected by LIBS-specific monoclonal antibodies (mAbs). Electronmicroscopy and crystal structures of the integrin in complex withvarious R(K)GD-like ligands support the theory that the integrinundergoes a major conformational change after or during ligand binding.

Currently two small molecule inhibitors of the αIIbβ3 exist: a cyclichomoarginine-glycine-aspartic acid peptide (eptifibatide) and an RGDpeptidomimetic (tirofiban). Both inhibitors act by competitivelyblocking the binding site for fibrinogen. Although both compounds havedemonstrated significant clinical benefit, tirofiban (Aggrastat™, Merckand Co., Inc.) is only available in a parenteral form and can causethrombocytopenia, dizziness and vasovagal reactions. Eptifibatide(Integrilin™, COR Therapeutics, Inc., Key Pharmaceuticals Inc.) is alsoonly available for parenteral administration and it too can causethrombocytopenia and hypotension. Crystal structure studies of theαIIbβ3 headpiece demonstrates that these inhibitors bind to both αIIband to the divalent cation in the β3 subunit's metal ion dependantadhesion site (MIDAS). It is believed that the interaction with theMIDAS metal ion induces conformational changes in the β3 which leads tothe increased the risk for thrombotic complications following αIIbβ3inhibitor therapy.

SUMMARY OF THE INVENTION

Previously, our scientists have identified inhibitors of αIIbβ3,particularly2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one,that are capable of inhibiting fibrinogen binding and plateletaggregation without inducing the binding of one or more integrin β3 LIBS-specific monoclonal antibodies (mAbs). These inhibitors are disclosedin U.S. patent application Ser. No. 12/514,286 (equivalent to U.S. Pub.No. 2010/0150913), the contents of which are hereby incorporated byreference in their entirety. Our scientists have now identified furtherinhibitors of αIIbβ3 that are capable of inhibiting fibrinogen bindingand platelet aggregation without inducing the binding of integrin β3LIBS. The present invention thus provides αIIbβ3 antagonists,pharmaceutical compositions comprising αIIbβ3 antagonists, new methodsof treatment and prophylaxis using αIIbβ3 antagonists, and new methodsto screen for αIIbβ3 antagonists that are capable of inhibitingfibrinogen binding without inducing β3 LIBS binding.

In the first aspect, the invention provides a compound of Formula P-I:

wherein:

-   -   i) A is S, N(H), CH₂, or O;    -   ii) R₁ is        -   phenyl optionally substituted with one or more nitro,            —C(O)N(R₅)(R₆) and/or —N(R₅)(R₆) and Y is a —C₁-C₄alkylene            (e.g., methylene) or arylene (e.g., phenylene);        -   phenyl substituted with one or more nitro and/or —N(R₅)(R₆)            and Y is a single bond;        -   phenyl substituted with —C(O)OR₃ and Y is a —C₁-C₄alkylene            (e.g., methylene) or arylene (e.g., phenylene);        -   heteroaryl (e.g., pyridyl, pyrazolyl, isoxazolyl, furyl,            thienyl) wherein said heteroaryl group is optionally            substituted with one or more —C₁-C₄alkyl (e.g., methyl) and            Y is a single bond, —C₁-C₄alkylene (e.g., methylene);        -   heteroaryl (e.g., pyridyl, pyrazolyl, thienyl or pyrimidyl)            wherein said heteroaryl group is substituted with halo            (e.g., fluoro), —C(O)OH, —CH₂C(O)OH, or —NH₂, and Y is            arylene (e.g., phenylene), for example R₁ is            (pyrazol-1-yl)-4-acetic acid (i.e.,            -(pyrazol-1-yl)-4—CH₂C(O)OH), thiophen-4-yl-2-carboxylic            acid (i.e., -(thiophen-4-yl)-2-C(O)OH), 2-fluoropyrid-4-yl            or 2-amino-pyrimid-5-yl, and Y is phenylene;        -   pyrazolyl, isoxazolyl, furyl or thienyl and Y is arylene            (e.g., phenylene) wherein said pyrazolyl, isoxazolyl, furyl            or thienyl is optionally substituted with one or more            —C₁-C₄alkyl (e.g., methyl), —C₀₋₄alkyl-C(O)OH, —N(R₁₃)(R₁₄),            or halo (e.g., fluoro); or        -   —C(O)N(R₄)(CH₂)₁₋₄—C(O)OR₃ (e.g., —C(O)N(H)CH₂C(O)OH) and Y            is a single bond, —C₁-C₄alkylene (e.g., methylene) or            arylene (e.g., phenylene),        -   —C(O)N(R₄)(CH₂)₁₋₄—N(R₁₃)(R₁₄) (e.g., —C(O)N(H)CH₂CH₂NH₂)            and Y is a single bond, —C₁-C₄alkylene (e.g., methylene) or            arylene (e.g., phenylene),        -   —N(R₄)—C(O)-heteroaryl (e.g., —N(R₄)—C(O)-pyridyl) wherein            said heteroaryl is optionally substituted with halo (e.g.,            fluoro) and Y is a single bond, —C₁-C₄alkylene (e.g.,            methylene) or arylene (e.g., phenylene),        -   —N(R₄)—C₁₋₄alkylene-heteroaryl (e.g.,            —N(R₄)—C₁₋₄alkylene-pyridyl) and Y is a single bond,            —C₁-C₄alkylene (e.g., methylene) or arylene (e.g.,            phenylene),        -   —N(R₁₀)—C(O)—[C(R₁₁)(R₁₂)]₁₋₄—N(R₁₃)(R₁₄), e.g.,            —N(R₁₀)—C(O)—[C(R₁₁)(R₁₂)]₂—N(R₁₃)(R₁₄) or            —N(R₁₀)—C(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄), and Y is a single            bond, —C₁-C₄alkylene (e.g., methylene) or arylene (e.g.,            phenylene), for example, R₁ is —N(H)—C(O)—CH₂CH₂—NH₂ or            —N(H)—C(O)—CH₂—NH₂ and Y is phenylene,        -   —N(R₁₀)—C(O)—C₃₋₁₀heterocycloalkyl (e.g.,            —N(R₁₀)—C(O)-piperidine) and Y is a single bond,            —C₁-C₄alkylene (e.g., methylene) or arylene (e.g.,            phenylene);        -   —N(R₄)(CH₂)₁₋₄—C(O)OR₄ (e.g., —N(H)CH₂C(O)OH) and Y is a            single bond, —C₁-C₄alkylene (e.g., methylene) or arylene            (e.g., phenylene);        -   —N(R₄)C(O)C(H)(NH₂)CH₂CH₂—C(O)OR₃ and Y is arylene (e.g.,            phenylene);        -   —N(R₄)C(O)C(H)(NH₂)CH₂-heteroaryl (e.g.,            —N(R₄)C(O)C(H)(NH₂)CH₂-thiazol-4-yl and Y is arylene (e.g.,            phenylene);        -   —N(R₄)C(O)C(H)(CH₃)—NH₂ and Y is arylene (e.g., phenylene);        -   —N(R₄)C(O)CH₂CH₂C(H)(NH₂)—COOH and Y is arylene (e.g.,            phenylene);        -   —N(R₄)C(O)—C(H)(NH₂)CH₂CH₂—COOH and Y is arylene (e.g.,            phenylene); or        -   —N(R₄)C(O)-heteroaryl (—N(H)C(O)isoxazolyl) and Y is arylene            (e.g., phenylene);    -   iii) R₂ is H, halo (e.g., fluoro) or —C₁-C₄alkyl (e.g., methyl);    -   iv) R_(a), R_(a)′, R_(b), R_(b)′, R_(c), R_(d), R_(d)′, R_(e),        and R_(e)′ are independently H or C₁-C₄alkyl (e.g., methyl or        ethyl);    -   V) R₃, R₄, R₅ and R₆ are independently H or C₁-C₄alkyl;    -   vi) R₁₀, R₁₁, R₁₂R₁₃ and R₁₄ are independently H or C₁₋₄alkyl        (e.g., methyl), in free or salt form.

In a further embodiment of the first aspect, the invention provides acompound of Formula P-I, wherein said compound is a compound of FormulaQ-I:

wherein:

-   -   i) A is S, N(H), CH₂, or O;    -   ii) R₁ is        -   phenyl optionally substituted with one or more nitro and/or            —N(R₅)(R₆) and Y is a —C₁-C₄alkylene (e.g., methylene) or            arylene (e.g., phenylene);        -   phenyl substituted with one or more nitro and/or —N(R₅)(R₆)            and Y is a single bond;        -   phenyl substituted with —C(O)OR₃ and Y is a —C₁-C₄alkylene            (e.g., methylene) or arylene (e.g., phenylene);        -   heteroaryl (e.g., pyridyl, pyrazolyl, isoxazolyl, furyl,            thienyl) wherein said heteroaryl group is optionally            substituted with one or more —C₁-C₄alkyl (e.g., methyl) and            Y is a single bond, —C₁-C₄alkylene (e.g., methylene);        -   pyrazolyl, isoxazolyl, furyl or thienyl and Y is arylene            (e.g., phenylene) wherein said pyrazolyl, isoxazolyl, furyl            or thienyl is optionally substituted with one or more            —C₁-C₄alkyl (e.g., methyl), —C₀₋₄alkyl-C(O)OH, —N(R₁₃)(R₁₄),            or halo (e.g., fluoro); or        -   —C(O)N(R₄)(CH₂)₁₋₄—C(O)OR₃ (e.g., —C(O)N(H)CH₂C(O)OH) and Y            is a single bond, —C₁-C₄alkylene (e.g., methylene) or            arylene (e.g., phenylene),        -   —C(O)N(R₄)(CH₂)₁₋₄—N(R₁₃)(R₁₄) (e.g., —C(O)N(H)CH₂CH₂NH₂)            and Y is a single bond, —C₁-C₄alkylene (e.g., methylene) or            arylene (e.g., phenylene),        -   —N(R₄)—C(O)-heteroaryl (e.g., —N(R₄)—C(O)-pyridyl) wherein            said heteroaryl is optionally substituted with halo (e.g.,            fluoro) and Y is a single bond, —C₁-C₄alkylene (e.g.,            methylene) or arylene (e.g., phenylene),        -   —N(R₄)—C₁₋₄alkylene-heteroaryl (e.g.,            —N(R₄)—C₁₋₄alkylene-pyridyl) and Y is a single bond,            —C₁-C₄alkylene (e.g., methylene) or arylene (e.g.,            phenylene),        -   —N(R₁₀)—C(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄) and Y is a single bond,            —C₁-C₄alkylene (e.g., methylene) or arylene (e.g.,            phenylene),        -   —N(R₁₀)—C(O)—C₃₋₁₀heterocycloalkyl (e.g.,            —N(R₁₀)—C(O)-piperidine) and Y is a single bond,            —C₁-C₄alkylene (e.g., methylene) or arylene (e.g.,            phenylene);        -   —N(R₄)(CH₂)₁₋₄—C(O)OR₄ (e.g., —N(H)CH₂C(O)OH) and Y is a            single bond, —C₁-C₄alkylene (e.g., methylene) or arylene            (e.g., phenylene);        -   —N(R₄)C(O)C(H)(NH₂)CH₂CH₂—C(O)OR₃ and Y is arylene (e.g.,            phenylene);        -   —N(R₄)C(O)C(H)(NH₂)CH₂-heteroaryl (e.g.,            —N(R₄)C(O)C(H)(NH₂)CH₂-thiazol-4-yl and Y is arylene (e.g.,            phenylene);        -   —N(R₄)C(O)C(H)(CH₃)—NH₂ and Y is arylene (e.g., phenylene);        -   —N(R₄)C(O)CH₂CH₂C(H)(NH₂)—COOH and Y is arylene (e.g.,            phenylene);        -   —N(R₄)C(O)—C(H)(NH₂)CH₂CH₂—COOH and Y is arylene (e.g.,            phenylene);        -   —N(R₄)C(O)-heteroaryl (—N(H)C(O)isoxazolyl) and Y is arylene            (e.g., phenylene);    -   iii) R₂ is H, halo (e.g., fluoro) or —C₁-C₄alkyl (e.g., methyl);    -   iv) R_(a), R_(a)′, R_(b), R_(b)′, R_(c), R_(d), R_(d)′, R_(e),        and R_(e)′ are independently H or C₁-C₄alkyl (e.g., methyl or        ethyl);    -   v) R₃, R₄, R₅ and R₆ are independently H or C₁-C₄alkyl;    -   vi) R₁₀, R₁₁, R₁₂R₁₃ and R₁₄ are independently H or C₁₋₄alkyl        (e.g., methyl), in free or salt form.

The invention further provides a compound of Formula P-I, as follows:

2.1. the compound of Formula P-I, wherein

-   -   R₁ is        -   phenyl optionally substituted with one or more nitro,            —C(O)N(R₅)(R₆) and/or —N(R₅)(R₆) and Y is a —C₁-C₄alkylene            (e.g., methylene) or arylene (e.g., phenylene);        -   phenyl substituted with one or more nitro and/or —N(R₅)(R₆)            and Y is a single bond;        -   phenyl substituted with —C(O)OR₃ and Y is a —C₁-C₄alkylene            (e.g., methylene) or arylene (e.g., phenylene);        -   heteroaryl (e.g., pyridyl, pyrazolyl, isoxazolyl, furyl,            thienyl) wherein said heteroaryl group is optionally            substituted with one or more —C₁-C₄alkyl (e.g., methyl) and            Y is a single bond, —C₁-C₄alkylene (e.g., methylene);        -   heteroaryl (e.g., pyridyl, pyrazolyl, thienyl or pyrimidyl)            wherein said heteroaryl group is substituted with halo            (e.g., fluoro), —C(O)OH, —CH₂C(O)OH, or —NH₂, and Y is            arylene (e.g., phenylene), for example R₁ is            (pyrazol-1-yl)-4-acetic acid (i.e.,            -(pyrazol-1-yl)-4-CH₂C(O)OH), thiophen-4-yl-2-carboxylic            acid (i.e., -(thiophen-4-yl)-2-C(O)OH), 2-fluoropyrid-4-yl            or 2-amino-pyrimid-5-yl, and Y is phenylene;        -   pyrazolyl, isoxazolyl, furyl or thienyl and Y is arylene            (e.g., phenylene) wherein said pyrazolyl, isoxazolyl, furyl            or thienyl is optionally substituted with one or more            —C₁-C₄alkyl (e.g., methyl), —C₀₋₄alkyl-C(O)OH, —N(R₁₃)(R₁₄),            or halo (e.g., fluoro);        -   —C(O)N(R₄)(CH₂)₁₋₄—C(O)OR₃ (e.g., —C(O)N(H)CH₂C(O)OH) and Y            is a single bond, —C₁-C₄alkylene (e.g., methylene) or            arylene (e.g., phenylene),        -   —C(O)N(R₄)(CH₂)₁₋₄—N(R₁₃)(R₁₄) (e.g., —C(O)N(H)CH₂CH₂NH₂)            and Y is a single bond, —C₁-C₄alkylene (e.g., methylene) or            arylene (e.g., phenylene),        -   —N(R₄)—C(O)-heteroaryl (e.g., —N(R₄)—C(O)-pyridyl) wherein            said heteroaryl is optionally substituted with halo (e.g.,            fluoro) and Y is a single bond, —C₁-C₄alkylene (e.g.,            methylene) or arylene (e.g., phenylene),        -   —N(R₄)—C₁₋₄alkylene-heteroaryl (e.g.,            —N(R₄)—C₁₋₄alkylene-pyridyl) and Y is a single bond,            —C₁-C₄alkylene (e.g., methylene) or arylene (e.g.,            phenylene),        -   —N(R₁₀)—C(O)—[C(R₁₁)(R₁₂)]₁₋₄—N(R₁₃)(R₁₄), e.g.,            —N(R₁₀)—C(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄), and Y is a single            bond, —C₁-C₄alkylene (e.g., methylene) or arylene (e.g.,            phenylene), for example, R₁ is N(H)—C(O)—CH₂CH₂—NH₂ and Y is            phenylene, —N(R₁₀)—C(O)—C₃₋₁₀heterocycloalkyl (e.g.,            —N(R₁₀)—C(O)-piperidine) and Y is a single bond,            —C₁-C₄alkylene (e.g., methylene) or arylene (e.g.,            phenylene);        -   —N(R₄)(CH₂)₁₋₄—C(O)OR₄ (e.g., —N(H)CH₂C(O)OH) and Y is a            single bond, —C₁-C₄alkylene (e.g., methylene) or arylene            (e.g., phenylene);        -   —N(R₄)C(O)C(H)(NH₂)CH₂CH₂—C(O)OR₃ and Y is arylene (e.g.,            phenylene);        -   —N(R₄)C(O)C(H)(NH₂)CH₂-heteroaryl (e.g.,            —N(R₄)C(O)C(H)(NH₂)CH₂-thiazol-4-yl and Y is arylene (e.g.,            phenylene);        -   —N(R₄)C(O)C(H)(CH₃)—NH₂ and Y is arylene (e.g., phenylene);        -   —N(R₄)C(O)CH₂CH₂C(H)(NH₂)—COOH and Y is arylene (e.g.,            phenylene);        -   —N(R₄)C(O)—C(H)(NH₂)CH₂CH₂—COOH and Y is arylene (e.g.,            phenylene);        -   —N(R₄)C(O)-heteroaryl (—N(H)C(O)isoxazolyl) and Y is arylene            (e.g., phenylene);        -   R₂ is H, halo (e.g., fluoro) or —C₁-C₄alkyl (e.g., methyl);        -   R_(a), R_(a)′, R_(b), R_(b)′, R_(c), R_(d), R_(d)′, R_(e),            and R_(e)′ are independently H or C₁-C₄alkyl (e.g., methyl            or ethyl);        -   R₃, R₄, R₅ and R₆ are independently H or C₁-C₄alkyl;        -   R₁₀, R₁₁, R₁₂R₁₃ and R₁₄ are independently H or C₁₋₄alkyl            (e.g., methyl),    -   2.2. the compound of Formula P-I, or 2.1, wherein R₁ is phenyl        optionally substituted with one or more nitro, —C(O)N(R₅)(R₆)        and/or —N(R₅)(R₆) and Y is a —C₁-C₄alkylene (e.g., methylene) or        arylene (e.g., phenylene);    -   2.3. the compound of Formula P-I, or 2.1 or 2.2, wherein R₁ is        phenyl optionally substituted with one or more nitro and/or        —N(R₅)(R₆) and Y is a —C₁-C₄alkylene (e.g., methylene) or        arylene (e.g., phenylene);    -   2.4. the compound of Formula P-I, or 2.1 or 2.2, wherein R₁ is        phenyl optionally substituted with —C(O)N(R₅)(R₆) and Y is a        —C₁-C₄alkylene (e.g., methylene) or arylene (e.g., phenylene);    -   2.5. the compound of Formula P-I, or 2.1, wherein R₁ is        heteroaryl (e.g., pyridyl, pyrazolyl, thienyl or pyrimidyl)        wherein said heteroaryl group is substituted with halo (e.g.,        fluoro), —C(O)OH, —CH₂C(O)OH, or —NH₂, and Y is arylene (e.g.,        phenylene), for example R₁ is (pyrazol-1-yl)-4-acetic acid        (i.e., -(pyrazol-1-yl)-4-CH₂C(O)OH), thiophen-4-yl-2-carboxylic        acid (i.e., -(thiophen-4-yl)-2-C(O)OH), 2-fluoropyrid-4-yl or        2-amino-pyrimid-5-yl, and Y is phenylene;    -   2.6. the compound of Formula P-I, 2.1 or 2.5, wherein R₁ is        pyridyl substituted with halo (e.g., fluoro) and Y is arylene        (e.g., phenylene), for example R₁ is 2-fluoropyrid-4-yl and Y is        phenylene;    -   2.7. the compound of Formula P-I, 2.1 or 2.5, wherein R₁ is        pyrimidyl substituted with NH₂, and Y is arylene (e.g.,        phenylene), for example R₁ is 2-amino-pyrimid-5-yl, and Y is        phenylene;    -   2.8. the compound of Formula P-I, or 2.1, wherein R₁ is        —N(R₁₀)—C(O)—[C(R₁₁)(R₁₂)]₁₋₄—N(R₁₃)(R₁₄), e.g.,        —N(R₁₀)—C(O)—[C(R₁₁)(R₁₂)]₂—N(R₁₃)(R₁₄) or        —N(R₁₀)—C(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄), and Y is a single bond,        —C₁-C₄alkylene (e.g., methylene) or arylene (e.g., phenylene),        for example, R₁ is N(H)—C(O)—CH₂CH₂—NH₂ or —N(H)—C(O)—CH₂—NH₂        and Y is phenylene;    -   2.9. the compound of Formula P-I, or 2.8, wherein R₁ is        —N(R₁₀)—C(O)—[C(R₁₁)(R₁₂)]₂—N(R₁₃)(R₁₄) or        —N(R₁₀)—C(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄), and Y is arylene (e.g.,        phenylene);    -   2.10. the compound of Formula P-I, or 2.9, wherein R₁ is        N(H)—C(O)—CH₂CH₂—NH₂;    -   2.11. the compound of Formula P-I, or 2.9, wherein R₁ is        N(H)—C(O)—CH₂—NH₂;    -   2.12. the compound of Formula P-I, or any of formulae 2.1-2.11,        wherein any of the substituents Y, R₁-R₁₄, R_(a), R_(a)′, R_(b),        R_(b)′, R_(c), R_(d), R_(d)′, R_(e), and R_(e)′, are        independently defined in any of formulae 3.1-3.51, or the        remaining substituents (i.e., substituents not yet defined in        any of the above formulae) are as defined in any of formulae        3.1-3.51;    -   2.13. the compound of Formula P-I, or any of formulae 2.1-2.12,        wherein said compound is selected from any of those disclosed in        any of formulae 3.45-13.50, and the following:

in free or salt form.

The invention further provides a compound of Formula Q-I as follows:

-   -   3.1 a Compounds of Formula Q-I, wherein A is S, N(H), CH₂, or O;    -   3.2 a Compound of Formula Q-I or 3.1, wherein A is O;    -   3.3 a Compound of Formula Q-I or 3.1, wherein A is S;    -   3.4 a Compound of Formula Q-I, or any of 3.1-3.3, wherein R₁ is        phenyl optionally substituted with one or more nitro and/or        —N(R₅)(R₆) and Y is a —C₁-C₄alkylene (e.g., methylene) or        arylene (e.g., phenylene);    -   3.5 a Compound of Formula Q-I or any of 3.1-3.4, wherein R₁ is        phenyl substituted with one or more nitro and/or —N(R₅)(R₆) and        Y is a single bond;    -   3.6 a Compound of Formula Q-I, or any of 3.1-3.4 wherein R₁ is        phenyl substituted with —C(O)OR₃ and Y is a —C₁-C₄alkylene        (e.g., methylene) or arylene (e.g., phenylene);    -   3.7 a Compound of Formula Q-I or any of 3.1-3.4, wherein R₁ is        heteroaryl (e.g., pyridyl, pyrazolyl, isoxazolyl, furyl,        thienyl) wherein said heteroaryl group is optionally substituted        with one or more —C₁-C₄alkyl (e.g., methyl) and Y is a single        bond or —C₁-C₄alkylene (e.g., methylene);    -   3.8 a Compound of Formula Q-I or any of 3.1-3.4, wherein R₁ is        pyrazolyl, isoxazolyl, furyl or thienyl and Y is arylene (e.g.,        phenylene) wherein pyrazolyl, isoxazolyl, furyl or thienyl is        optionally substituted with one or more —C₁-C₄alkyl (e.g.,        methyl) or halo (e.g., fluoro);    -   3.9 a Compound of Formula Q-I or 3.8, wherein R₁ is pyrazolyl        and Y is arylene (e.g., phenylene) wherein said pyrazolyl is        optionally substituted with one or more —C₁-C₄alkyl (e.g.,        methyl) or halo (e.g., fluoro);    -   3.10 a Compound of Formula Q-I or or 3.8 or 3.9, wherein R₁ is        pyrazolyl and Y is phenylene wherein said pyrazolyl is        optionally substituted with one or more —C₁-C₄alkyl (e.g.,        methyl) or halo (e.g., fluoro);    -   3.11 formula 3.10, wherein said pyrazolyl is substituted on the        meta-position of the phenylene (e.g., relative to the point of        attachment);    -   3.12 a Compound of Formula Q-I or any of 3.1-3.4, wherein R₁ is        —C(O)N(R₄)(CH₂)₁₋₄—C(O)OR₃ (e.g., —C(O)N(H)CH₂C(O)OH),        —C(O)N(R₄)(CH₂)₁₋₄—N(R₁₃)(R₁₄) (e.g., —C(O)N(H)CH₂CH₂NH₂),        —N(R₄)—C(O)-heteroaryl (e.g., —N(R₄)—C(O)-pyridyl),        —N(R₄)—C₁₋₄alkylene-heteroaryl (e.g.,        —N(R₄)—C₁₋₄alkylene-pyridyl),        —N(R₁₀)—C(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄),        —N(R₁₀—C(O)—C₃₋₁₀heterocycloalkyl (e.g.,        —N(R₁₀)—C(O)-piperidine) or —N(R₄)(CH₂)₁₋₄—C(O)OR₄ (e.g.,        —N(H)CH₂C(O)OH) and Y is a single bond, —C₁-C₄alkylene (e.g.,        methylene) or arylene (e.g., phenylene);    -   3.13 a Compound of Formula Q-I or 3.12, wherein Y is an arylene        (e.g., phenylene);    -   3.14 a Compound of Formula Q-I or any of 3.1-3.4, wherein:        -   R₁ is            -   phenyl optionally substituted with one or more nitro                and/or —N(R₅)(R₆),            -   phenyl substituted with —C(O)OR₃,            -   pyrazolyl, isoxazolyl, furyl or thienyl wherein said                pyrazolyl, isoxazolyl, furyl or thienyl is optionally                substituted with one or more —C₁-C₄alkyl (e.g., methyl),                —C₀₋₄alkyl-C(O)OH, —N(R₁₃)(R₁₄), or halo (e.g., fluoro),            -   —C(O)N(R₄)(CH₂)₁₋₄—C(O)OR₃ (e.g., —C(O)N(H)CH₂C(O)OH),            -   —C(O)N(R₄)(CH₂)₁₋₄—N(R₁₃)(R₁₄) (e.g.,                —C(O)N(H)CH₂CH₂NH₂),            -   —N(R₄)—C(O)-heteroaryl (e.g., —N(R₄)—C(O)-pyridyl)                wherein said heteroaryl is optionally substituted with                halo (e.g., fluoro),            -   —N(R₄)—C₁₋₄alkylene-heteroaryl (e.g.,                —N(R₄)—C₁₋₄alkylene-pyridyl),            -   —N(R₁₀)—C(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄),            -   —N(R₁₀)—C(O)—C₃₋₁₀heterocycloalkyl (e.g.,                —N(R₁₀)—C(O)-piperidine),            -   —N(R₄)(CH₂)₁₋₄—C(O)OR₄ (e.g., —N(H)CH₂C(O)OH);            -   —N(R₄)C(O)C(H)(NH₂)CH₂CH₂—C(O)OR₃;            -   —N(R₄)C(O)C(H)(NH₂)CH₂-heteroaryl (e.g.,                —N(R₄)C(O)C(H)(NH₂)CH₂-thiazol-4-yl and Y is arylene                (e.g., phenylene);            -   —N(R₄)C(O)C(H)(CH₃)—NH₂;            -   —N(R₄)C(O)CH₂CH₂C(H)(NH₂)—COOH,            -   —N(R₄)C(O)—C(H)(NH₂)CH₂CH₂—COOH or,            -   —N(R₄)C(O)-heteroaryl (—N(H)C(O)isoxazolyl),        -   and Y is arylene (e.g., phenylene);    -   3.15 a Compound of Formula Q-I or 3.12, 3.13 or 3.14, wherein Y        is a phenylene;    -   3.16 formula 3.15, wherein R₁ is substituted on the        meta-position of the phenylene (e.g., relative to the point of        attachment);    -   3.17 a Compound of Formula Q-I or any of 3.12-3.16, wherein R₁        is —N(R₁₀)—C(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄);    -   3.18 a Compound of Formula Q-I or any of 3.12-3.16, wherein R₁        is —N(R₁₀)—C(O)—C₃₋₁₀heterocycloalkyl (e.g.,        —N(R₁₀)—C(O)-piperidine);    -   3.19 a Compound of Formula Q-I or any of 3.12-3.18, wherein R₁₀,        is H or C₁₋₄alkyl (e.g., methyl);    -   3.20 a Compound of Formula Q-I or any of 3.12-3.18, wherein R₁₀        is H,    -   3.21 a Compound of Formula Q-I or any of 3.12-3.18, wherein R₁₀        is C₁₋₄alkyl (e.g., methyl);    -   3.22 a Compound of Formula Q-I or any of 3.12-3.17 or 3.19-3.21,        wherein R₁₁, R₁₂R₁₃ and R₁₄ are independently H or C₁₋₄alkyl        (e.g., methyl);    -   3.23 a Compound of Formula Q-I or any of 3.12-3.17 or 3.19-3.21,        wherein R₁₁ and R₁₂ are both H;    -   3.24 a Compound of Formula Q-I or any of 3.12-3.17 or 3.19-3.21,        wherein R₁₁ is H and R₁₂ is C₁₋₄alkyl (e.g., methyl);    -   3.25 a Compound of Formula Q-I or any of 3.12-3.17 or 3.19-3.21,        wherein R₁₃ and R₁₄ are independently H or C₁₋₄alkyl (e.g.,        methyl);    -   3.26 a Compound of Formula Q-I or any of 3.12-3.17 or 3.19-3.21,        wherein R₁₃ and R₁₄ are both H,    -   3.27 a Compound of Formula Q-I or any of 3.12-3.17 or 3.19-3.21,        wherein R₁₃ is H and R₁₄ is C₁₋₄alkyl (e.g., methyl);    -   3.28 a Compound of Formula Q-I or any of 3.1-3.27, wherein R₂ is        H, halo (e.g., fluoro) or C₁-C₄alkyl (e.g., methyl);    -   3.29 a Compound of Formula Q-I or any of 3.1-3.28, wherein R₂ is        —C₁-C₄alkyl (e.g., methyl);    -   3.30 a Compound of Formula Q-I or any of 3.1-3.27, wherein R₂ is        H or halo (e.g., fluoro);    -   3.31 a Compound of Formula Q-I or any of 3.1-3.27, wherein R₂ is        H;    -   3.32 a Compound of Formula Q-I or any of 3.1-3.27, wherein R₂ is        halo (e.g., fluoro);    -   3.33 a Compound of Formula Q-I or any of 3.1-3.32, wherein        R_(a), R_(a)′, R_(b), R_(b)′, R_(c), R_(d), R_(d)′, R_(e), and        R_(e)′ are independently H or C₁-C₄alkyl (e.g., methyl or        ethyl);    -   3.34 a Compound of Formula Q-I or any of 3.1-3.32, wherein        R_(a)′, R_(b)′, R_(d)′ and R_(e)′ are H,    -   3.35 a Compound of Formula Q-I or any of 3.1-3.34, wherein        R_(a), R_(b), R_(c), R_(d) and R_(e) are independently H or        C₁-C₄alkyl (e.g., methyl or ethyl);    -   3.36 a Compound of Formula Q-I or any of 3.1-3.34, wherein        R_(a), R_(b), R_(c), R_(d) and R_(e) are independently H,    -   3.37 a Compound of Formula Q-I or any of 3.1-3.34, wherein        R_(a), R_(b), R_(c), R_(d) and R_(e) are independently        C₁-C₄alkyl (e.g., methyl or ethyl);    -   3.38 a Compound of Formula Q-I or any of 3.1-3.37, wherein R₃,        R₄, R₅ and R₆ are independently H or C₁-C₄alkyl;    -   3.39 a Compound of Formula Q-I or any of 3.1-3.37, wherein R₃,        R₄, R₅ and R₆ are independently H;    -   3.40 a Compound of Formula Q-I or any of 3.1-3.37, wherein R₃,        R₄, R₅ and R₆ are independently C₁-C₄alkyl;    -   3.41 a Compound of Formula Q-I or any of 3.1-3.40, wherein the        compound is as shown below:

-   -   wherein        -   R₁ is:            -   phenyl optionally substituted with one or more nitro                and/or —N(R₅)(R₆); phenyl substituted with —C(O)OR₃;            -   pyrazolyl optionally substituted with one or more                —C₁-C₄alkyl (e.g., methyl) or halo (e.g., fluoro);            -   —C(O)N(R₄)(CH₂)₁₋₄—C(O)OR₃ (e.g., —C(O)N(H)CH₂C(O)OH);            -   —C(O)N(R₄)(CH₂)₁₋₄—N(R₁₃)(R₁₄) (e.g.,                —C(O)N(H)CH₂CH₂NH₂);            -   —N(R₄)—C(O)-heteroaryl (e.g., —N(R₄)—C(O)-pyridyl);            -   —N(R₄)—C₁₋₄alkylene-heteroaryl (e.g.,                —N(R₄)—C₁₋₄alkylene-pyridyl);            -   —N(R₁₀)—C(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄);            -   —N(R₁₀)—C(O)—C₃₋₁₀heterocycloalkyl (e.g.,                —N(R₁₀)—C(O)-piperidine);            -   —N(R₄)(CH₂)₁₋₄—C(O)OR₄ (e.g., —N(H)CH₂C(O)OH);            -   —N(R₄)C(O)C(H)(NH₂)CH₂CH₂—C(O)OR₃;            -   —N(R₄)C(O)C(H)(NH₂)CH₂-heteroaryl (e.g.,                —N(R₄)C(O)C(H)(NH₂)CH₂-thiazol-4-yl;            -   —N(R₄)C(O)C(H)(CH₃)—NH₂;            -   —N(R₄)C(O)CH₂CH₂C(H)(NH₂)—COOH;            -   —N(R₄)C(O)—C(H)(NH₂)CH₂CH₂—COOH;            -   —N(R₄)C(O)-heteroaryl (—N(H)C(O)isoxazolyl);        -   R₂ is H, halo (e.g., fluoro) or —C₁-C₄alkyl (e.g., methyl);        -   R₃, R₄, R₅ and R₆ are independently H or C₁-C₄alkyl;        -   R₁₀, R₁₁, R₁₂R₁₃ and R₁₄ are independently H or C₁₋₄alkyl            (e.g., methyl);        -   R_(a), R_(a)′, R_(b), R_(b)′, R_(c), R_(d), R_(d)′, R_(e),            and R_(e)′ are H,    -   3.42 a Compound of Formula Q-I or any of 3.1-3.40, wherein the        compound is as shown below:

-   -   wherein        -   R₁ is:            -   phenyl optionally substituted with one or more nitro                and/or —N(R₅)(R₆);            -   phenyl substituted with —C(O)OR₃;            -   pyrazolyl optionally substituted with one or more                —C₁-C₄alkyl (e.g., methyl) or halo (e.g., fluoro);            -   C(O)N(R₄)(CH₂)₁₋₄—C(O)OR₃ (e.g., —C(O)N(H)CH₂C(O)OH);            -   —N(R₄)—C(O)-heteroaryl (e.g., —N(R₄)—C(O)-pyridyl);                —N(R₄)—C₁₋₄alkylene-heteroaryl (e.g.,                —N(R₄)—C₁₋₄alkylene-pyridyl);            -   —N(R₁₀)—C(O)—C(R₁₁)(R₁₂)—NR₁₃)(R₁₄);            -   —N(R₁₀)—C(O)—C₃₋₁₀heterocycloalkyl (e.g.,                —N(R₁₀)—C(O)-piperidine); or            -   —N(R₄)(CH₂)₁₋₄—C(O)OR₄ (e.g., —N(H)CH₂C(O)OH);        -   R₂ is H, halo (e.g., fluoro) or —C₁-C₄alkyl (e.g., methyl);        -   R₃, R₄, R₅ and R₆ are independently H or C₁-C₄alkyl;        -   R₁₀, R₁₁, R₁₂R₁₃ and R₁₄ are independently H or C₁₋₄alkyl            (e.g., methyl);        -   R_(a), R_(a)′, R_(b), R_(b)′, R_(c), R_(d), R_(d)′, R_(e),            and R_(e)′ are independently H or C₁-C₄alkyl (e.g., methyl            or ethyl);    -   3.43 a Compound of Formula Q-I or any of 3.1-3.41, wherein the        compound is as shown below:

-   -   wherein        -   R₁ is —N(R₁₀)—C(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄), e.g.,            —NHC(O)CH₂NH₂;        -   R₂ is H or halo (e.g., fluoro);        -   R_(a), R_(a)′, R_(b), R_(b)′, R_(c), R_(d), R_(d)′, R_(e),            and R_(e)′ are independently H or C₁-C₄alkyl (e.g., methyl            or ethyl);        -   R₁₀, R₁₁, R₁₂R₁₃ and R₁₄ are independently H or C₁₋₄alkyl            (e.g., methyl);    -   3.44 any of the preceding formulae 3.1-3.43, wherein the        Compound of Formula Q-I is selected from any of the following:

-   -   3.45 any of formulae 3.1-3.44, wherein the Compound of Formula        Q-I is selected from any of the following:

-   -   3.46 any of formulae 3.1-3.44 wherein the Compound of Formula        Q-I is selected from any of the following:

-   -   3.47 any of formulae 3.1-3.44 wherein the Compound of Formula        Q-I is selected from any of the following:

-   -   3.48 any of formulae 3.1-3.44 wherein the Compound of Formula        Q-I is:

-   -   3.49 any of formulae 3.1-3.44 wherein the Compound of Formula        Q-I is:

-   -   3.50 any of formulae 3.1-3.44 wherein the Compound of Formula        Q-I is:

-   -   3.51 any of the preceding formulae wherein the compound of        Formula Q-I has an IC₅₀ value of less than 100 μM, preferably        less than 75 μM, more preferably less than 50 μM, most        preferably, less than 25 μM in an aggregation assay as described        in Example 44 and/or a percentage of inhibition of greater than        30%, preferably greater than 40%, more preferably, greater than        50%, more preferably, greater than 60%, most preferably greater        than 70% at a concentration of 100 μM or less in an adhesion        assay as described in Example 44.    -   in free or salt form.

In a further embodiment of the first aspect, the invention provides aCompound of Formula P-I or Q-I, preferably formula 3.43, wherein:

-   -   R₁ is —N(R₁₀)—C(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄), e.g., —NHC(O)CH₂NH₂;    -   R₂ is H;    -   R_(a), R_(a)′, R_(b), R_(b)′, R_(c), R_(d), R_(d)′, R_(e) and        R_(e)′ are independently H or C₁-C₄alkyl (e.g., methyl or        ethyl);    -   R₁₀, R₁₁, R₁₂R₁₃ and R₁₄ are independently H or C₁₋₄alkyl (e.g.,        methyl),    -   in free or salt form.

In a further embodiment of the first aspect, the invention provides aCompound of Formula P-I or Q-I, preferably formula 3.43, wherein:

-   -   R₁ is —N(R₁₀)—C(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄), e.g., —NHC(O)CH₂NH₂;    -   R₂ is H;    -   R_(a), R_(a)′, R_(b), R_(b)′, R_(c), R_(d), R_(d)′, R_(e) and        R_(e)′ are H;    -   R₁₀, R₁₁, R₁₂R₁₃ and R₁₄ are independently H or C₁₋₄alkyl (e.g.,        methyl),    -   in free or salt form.

In the second aspect, the Invention provides a Compound of Formula I-Aselected from:

wherein R₂ is H, halo (e.g., fluoro) or C₁-C₄-alkyl (e.g., methyl), infree or salt form. In a particular embodiment, the Compound of FormulaI-A is the compound having the para-substituted carboxylic acid and R₂is H.

In the third aspect, the Invention provides a Compound of Formula I-Bselected from:

wherein R₂ is H, halo (e.g., fluoro) or C₁-C₄-alkyl (e.g., methyl), infree or salt form.

In the fourth aspect, the invention provides a Compound of Formula I-Cselected from any one of the following:

in free or salt form.

Preferably, the Compounds of the Present Invention (e.g., any of thecompounds hereinbefore described, e.g. any of Compound of Formula P-I,any of 2.1-2.13, Formula Q-I, e.g., any of 3.1-3.51, or the compounddescribed in paragraph [0021] or [0022], any of the Compound of FormulaI-A, I-B or I-C, as hereinbefore described, in free or salt form) areselected from the Compounds of Formula Q-I, e.g., any of 3.1-3.51, morepreferably, the compounds of formula 3.45, most preferably formula 3.49,in free or salt form.

In the fifth aspect, the invention provides to a PharmaceuticalComposition (Composition Q-I) comprising a Compound of the Inventionhereinbefore described, e.g., a Compound of Formula P-I, any of2.1-2.13, Formula Q-I, e.g., any of 3.1-3.51, or the compound describedin paragraph [0021] or [0022], any of the Compound of Formula I-A, I-Bor I-C, preferably selected from the Compounds of Formula Q-I, e.g., anyof 3.1-3.51, more preferably, the compounds of formula 3.45, mostpreferably 3.49, as hereinbefore described, in free or pharmaceuticallyacceptable salt form, in combination or association with apharmaceutically acceptable diluent or carrier. Composition Q ashereinbefore described is useful, e.g., for preventing or inhibitingplatelet adhesion and/or aggregation in treating thrombotic a disorderin a subject in need thereof. In a particular embodiment, the inventionprovides Composition Q-I as hereinbefore described, wherein the compoundis a Compound of Formula Q-I, any of 3.1-3.51, or the compound describedin paragraph [0021] or [0022], more preferably, the compounds of formula3.45, most preferably 3.49, in free or pharmaceutically acceptable saltform. In yet another embodiment, the invention provides a PharmaceuticalComposition Q-I as hereinbefore described useful for inhibiting orreducing platelet aggregation and/or adhesion.

In the sixth aspect, the invention provides a method for inhibiting orreducing platelet aggregation and/or adhesion comprising administeringto a subject in need thereof, an effective amount of a Compound ofFormula P-I, any of 2.1-2.13, Formula Q-I, e.g., any of 3.1-3.51, or thecompound described in paragraph [0021] or [0022] any of the Compound ofFormula I-A, I-B or I-C, preferably a Compound of Formula Q-I, e.g., anyof 3.1-3.51, or the compound described in paragraph [0021] or [0022],more preferably, a compound of formula 3.45, most preferably 3.49, infree or pharmaceutically acceptable salt form, such that plateletaggregation and/or adhesion is reduced (Method Q-I).

The invention further provides for the following methods:

-   -   6.1 Method Q-I, wherein the compound is a Compound of Formula        P-I, any of 2.1-2.13, Formula Q-I, e.g., any of 3.1-3.51, or the        compound described in paragraph [0021] or [0022], more        preferably, the compound of formula 3.45, most preferably 3.49,        in free or pharmaceutically acceptable salt form;    -   6.2 Method Q-I or 6.1, wherein reduction of platelet aggregation        and/or adhesion treats or inhibits a thrombotic disorder, e.g.        selected from a group consisting of stroke, myocardial        infarction, unstable angina, abrupt closure following        angioplasty or stent placement, thrombosis induced by peripheral        vascular surgery, peripheral vascular disease or thrombotic        disorders resulting from atrial fibrillation or inflammation.

In a particular embodiment, the invention provides Method Q-I, e.g.,formula 6.1 or 6.2, wherein both platelet aggregation and adhesion arereduced (Method Q-I′).

In the seventh aspect, the invention provides a method for the treatmentor prophylaxis of a thrombotic disorder comprising administering to asubject at risk of a thrombotic disorder, an effective amount of acompound of Formula P-I, any of 2.1-2.13, Formula Q-I, e.g., any of3.1-3.51, or the compound described in paragraph [0021] or [0022], anyof the Compound of Formula I-A, I-B or I-C, as hereinbefore described,in free or pharmaceutically acceptable salt form, such that plateletaggregation and/or adhesion is reduced (Method Q-II).

The invention further provides for the following methods:

-   -   7.1 Method Q-II, wherein said thrombotic disorder is selected        from a group consisting of stroke, myocardial infarction,        unstable angina, abrupt closure following angioplasty or stent        placement, thrombosis induced by peripheral vascular surgery,        peripheral vascular disease or thrombotic disorders resulting        from atrial fibrillation or inflammation;    -   7.2 Method Q-II or 7.1, wherein said thrombotic disorder is        thrombosis as a result of angioplasty or stent placement;    -   7.3 Method Q-II, 7.1 or 7.2, wherein subject at risk of        thrombotic disorder is a subject who has a history of vascular        surgery;    -   7.4 Method Q-II or any of Methods 7.1-7.3, further comprises        administering to said subject an effective amount of at least        one therapeutic agent selected from a group consisting of        anti-coagulant, antiplatelet, and thrombolytic agents in        conjunction with a Compound of Formula P-I, any of 2.1-2.13,        Formula Q-I, e.g., any of 3.1-3.51, or the compound described in        paragraph [0021] or [0022], any of the Compound of Formula I-A,        I-B or I-C, as hereinbefore described, in free or        pharmaceutically acceptable salt form;    -   7.5 Method Q-II, or any of Methods 7.1-7.4, further comprises        administering to said subject an effective amount of at least        one therapeutic agent selected from a group consisting of        heparin, low molecular weight heparins, bivalirudin,        Fondaparinux, warfarin, Acenocoumarol, Phenprocoumon,        Phenindione, Abbokinase (urokinase), streptokinase, alteplase,        retaplase, tenecteplase, prasugrel, aspirin, ticlopidine,        clopidogrel, abciximab, eptifibatide and tirofiban in        conjunction with a Compound of Formula P-I, any of 2.1-2.13,        Formula Q-I, e.g., any of 3.1-3.51, or the compound described in        paragraph [0021] or [0022], any of the Compound of Formula I-A,        I-B or I-C, as hereinbefore described, in free or        pharmaceutically acceptable salt form;    -   7.6 Method Q-II or any of Methods 7.1-7.4, further comprises        administering to said subject an anticoagulant or thrombolytic        agent in conjunction with a Compound of Formula P-I, any of        2.1-2.13, Formula Q-I, e.g., any of 3.1-3.51, or the compound        described in paragraph [0021] or [0022], any of the Compound of        Formula I-A, I-B or I-C, as hereinbefore described, in free or        pharmaceutically acceptable salt form;    -   7.7 Method Q-II or any of Methods 7.1-7.4, further comprises        administering to said subject an effective amount of heparin in        conjunction with a Compound of Formula P-I, any of 2.1-2.13,        Formula Q-I, e.g., any of 3.1-3.51, or the compound described in        paragraph [0021] or [0022], any of the Compound of Formula I-A,        I-B or I-C, as hereinbefore described, in free or        pharmaceutically acceptable salt form.    -   7.8 Method Q-II or any of Methods 7.1-7.7, wherein the Compound        of the Invention is a Compound of Formula P-I, any of 2.1-2.13,        Formula Q-I, e.g., any of 3.1-3.51, or the compound described in        paragraph [0021] or [0022], more preferably, the compounds of        formula 3.45, most preferably formula 3.49, in free or        pharmaceutically acceptable salt form.

The invention further provides any of the foregoing methods wherein thecompounds of the present invention (a) reduce platelet inhibition with apercentage of inhibition of greater than 30%, preferably greater than50% at a concentration of 100 μM or less; and/or (b) reduce plateletaggregation, e.g., with an IC₅₀ of less than 100 μM, preferably lessthan 25 μM in an ADP or other agonist-induced platelet aggregation assayand/or in a fibrinogen binding assay as described in the examples below.

In a preferred embodiment, the invention is a method for the treatmentor prophylaxis of a thrombotic disorder comprising administering aneffective amount of a Compound of Formula P-I, any of 2.1-2.13, FormulaQ-I, any of 3.1-3.51, or the compound described in paragraph [0021] or[0022], more preferably, the compounds of formula 3.44, most preferably3.49 in free or pharmaceutically acceptable salt form.

In a particular embodiment, the invention is a method for the treatmentor prophylaxis of a thrombotic disorder comprising administering heparinin conjunction with the Compound of Formula P-I, any of 2.1-2.13,Formula Q-I, e.g., any of 3.1-3.51, or the compound described inparagraph [0021] or [0022], any of the Compound of Formula I-A, I-B orI-C, as hereinbefore described, in free or pharmaceutically acceptablesalt form, preferably a Compound of Formula P-I, any of 2.1-2.13,Formula Q-I, any of 3.1-3.51, or the compound described in paragraph[0021] or [0022], more preferably, the compounds of formula 3.44, mostpreferably 3.49 in free or pharmaceutically acceptable salt form.

Without being bound to any theory, it is believed that binding of ligandby the receptor induces conformational changes in αIIbβ3, exposing theligand-induced binding sites (LIBS). With traditional αIIbβ3-inhibitorssuch as tirofiban and eptifibatide, binding of these compounds to boththe αIIb and to the divalent cation in the β3 subunit's metal iondependant adhesion site (MIDAS) inhibits platelet adhesion. It isbelieved, however, that the interaction with the β3 subunit's metal iondependant adhesion site (MIDAS) is likely to be responsible forinitiating the conformational change which result in both thethrombocytopenia and the increased mortality rate of traditional αIIbβ3antagonists. The present invention identifies αIIbβ3 inhibitors that arecapable of inhibiting fibrinogen binding without inducing the binding ofone more integrin β3 LIBS-specific mAbs. Therefore, in one embodiment,the Compounds of the Invention e.g., the Compound of Formula P-I, any of2.1-2.13, Formula Q-I, e.g., any of 3.1-3.51, or the compound describedin paragraph [0021] or [0022], any of the Compound of Formula I-A, I-Bor I-C, preferably, the compounds of formula 3.45, most preferablyformula 3.49, in free or salt form may bind to αIIb, and in some casesinduce αIIb LIBS exposure, without inducing β3 LIBS exposure. Suchcompounds thus demonstrate specific binding to αIIbβ3 integrin andinhibition of platelet adhesion without the disadvantage of inducing thechange in conformation of the β3 and consequent risk of complicationsfollowing dissociation of the compounds from the αIIbβ3.

In the eighth aspect, the invention provides a drug-eluting stentwherein the drug or drugs eluted comprise a Platelet Inhibitor of theInvention, or a Compound of P-I, any of 2.1-2.13, Formula Q-I, e.g., anyof 3.1-3.51, or the compound described in paragraph [0021] or [0022],any of the Compound of Formula I-A, I-B or I-C, preferably, the Compoundof Formula Q-I, more preferably, the compounds of formula 3.45, mostpreferably formula 3.49, in free or pharmaceutically acceptable saltform as hereinbefore described. For example, the invention provides astent, e.g., an arterial stent, for example a coronary artery or carotidartery stent, which comprises a biocompatible polymer matrix whichcomprises or is associated with a Compound of P-I, any of 2.1-2.13,Formula Q-I, e.g., any of 3.1-3.51, or the compound described inparagraph [0021] or [0022], any of the Compound of Formula I-A, I-B orI-C, in free or pharmaceutically acceptable salt form as hereinbeforedescribed. The stent may be made of metal, plastic, biodegradable orbioabsorbable material or combination thereof, e.g., stainless steel,nickel-titanium alloy, colbalt-alloy, tantalum, silicone,polytetrafluoroethylene, magnesium alloy or poly-L-lactide. For example,a stent may be a metallic stent (e.g., stainless steel, nickel-titaniumalloy, colbalt alloy, or tantalum) partially or wholly coated with abiocompatible polymer, e.g., a plastic (e.g., polytetrafluoroethylene)or a polymeric carrier (e.g., phosphorylcholine or polylactic acid)which polymer comprises or is associated with a Compound of P-I, any of2.1-2.13, Formula Q-I, e.g., any of 3.1-3.51, or the compound describedin paragraph [0021] or [0022], any of the Compound of Formula I-A, I-Bor I-C, preferably, the compounds of formula Q-I, more preferably,formula 3.45, most preferably formula 3.49, in free or pharmaceuticallyacceptable salt form as hereinbefore described, e.g., such that saidCompound is presented or released in a manner and amount effective toinhibit platelet adhesion and/or aggregation in the vicinity of thestent. The stent may further comprise or be associated with anadditional drug or drugs, e.g., an antiproliferative agent, e.g.,sirolimus, everolimus, zotarolimus, tacrolimus, or paclitaxel, and/or ananticoagulant, e.g., heparin.

In the nineth aspect, the invention provides a Compound of Formula P-I,any of 2.1-2.13, Formula Q-I, e.g., any of 3.1-3.51, or the compounddescribed in paragraph [0021] or [0022], any of the Compound of FormulaI-A, I-B or I-C, preferably, Compounds of Formula P-I, any of 2.1-2.13,Formula Q-I, e.g., any of 3.1-3.51, or the compound described inparagraph [0021] or [0022], more preferably, the compounds of formula3.45, most preferably formula 3.49, in free or pharmaceuticallyacceptable salt form as hereinbefore described, for use as apharmaceutical, e.g. use of a Compound of Formula P-I, any of 2.1-2.13,Formula Q-I, e.g., any of 3.1-3.51, or the compound described inparagraph [0021] or [0022], any of the Compound of Formula I-A, I-B orI-C, preferably Compounds of Formula P-I, any of 2.1-2.13, Formula Q-I,e.g., any of 3.1-3.51, or the compound described in paragraph [0021] or[0022], more preferably, the compounds of formula 3.45, most preferablyformula 3.49, in free or pharmaceutically acceptable salt form ashereinbefore described, e.g., (in the manufacture of a medicament) forthe treatment or prophylaxis of a thrombotic disorder, e.g., accordingto any of Method Q-I, e.g., formula 6.1 or 6.2, or Method Q-II or any ofmethods 7.1-7.8.

In the tenth aspect, the invention provides a Pharmaceutical Compositioncomprising a Compound of Formula P-I, any of 2.1-2.13, Formula Q-I,e.g., any of 3.1-3.51, or the compound described in paragraph [0021] or[0022], any of the Compound of Formula I-A, I-B or I-C, preferably,Compounds of Formula P-I, any of 2.1-2.13, Formula Q-I, e.g., any of3.1-3.51, or the compound described in paragraph [0021] or [0022], morepreferably, the compounds of formula 3.45, most preferably formula 3.49,in free or pharmaceutically acceptable salt form as hereinbeforedescribed, for use as a pharmaceutical e.g., (in the manufacture of amedicament) for the treatment or prophylaxis of a thrombotic disorder,e.g., according to any of Method Q-I, e.g., formula 6.1 or 6.2, orMethod Q-II or any of methods 7.1-7.8.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “αIIbβ3” or “integrin αIIbβ3” refers to thereceptor on the surface of human platelets. It is a heterodimericcomplex composed of both αIIb and β3 subunits responsible for bindingadhesive plasma proteins, most notably fibrinogen and von Willebrandfactor.

The term “antagonist” refers to any ligand or molecule that binds toreceptors and competitively or noncompetitively blocks the binding ofligand to that receptor. Therefore, “αIIbβ3 antagonist” refers to anyligand or molecule that competitively or noncompetitively blocks αIIbβ3.

“LIBS” refers to ligand-induced binding sites on αIIbβ3 that arepresented or exposed upon the binding of a ligand or antagonist by thereceptor.

“LIBS-specific mAbs” refers to monoclonal antibodies that bind to theexposed ligand-induced binding sites of αIIbβ3. Examples ofLIBS-specific mAbs include APS, PMI-1 and LIBS1.

The term “thrombotic disorders” refers to disorders characterized byformation of a thrombus that obstructs vascular blood flow. Examples ofthrombotic disorders include stroke, myocardial infarction, stable orunstable angina, peripheral vascular disease, abrupt closure followingangioplasty or stent placement and thrombosis induced by vascularsurgery. Thrombotic disorders also include disorders characterized byformation of a thrombus caused by atrial fibrillation or inflammation.

The phrase “subject at risk of thrombotic disorders” or “subject in needthereof” includes subjects who have a history of vascular intervention(e.g. angioplasty, stent placement, aortocoronary bypass or insertion ofprosthetic heart valves), cardiovascular abnormality (e.g. atrialfibrillation) or a family history of vascular diseases (e.g., coronaryartery disease (CAD), systemic hypertension, diabetes mellitus,hyperlipidemia, bicuspid aortic valve, hypertrophic cardiomyopathy ormitral valve prolapse). The term “subject” may include human ornon-human (e.g., an animal).

The term “platelet adhesion” refers to the binding of platelet membraneproteins to fibrinogen, collagen, von Willebrand factor (vWF) or otheradhesive glycoproteins (e.g., fibronectin, laminin).

The term “platelet aggregation” refers to the attachment of activatedplatelets one to another, which results in the formation of aggregatesor clumps of activated platelets.

The phrase “inhibit or reduce platelet adhesion and/or aggregation” isintended to mean at least a 30% inhibition of platelet activity at aconcentration of 100 μM or lower in a given assay, relative to plateletactivity in the absence of the compound.

The phrase “antagonist known to expose β3 LIBS” herein refers to agentsthat induce conformational in β3, for example tirofiban.

The term “anticoagulants” herein refers to any compound or substancethat either stimulates natural inhibitor of coagulant proteases orblocks the coagulation cascade. Examples of anticoagulants include, butare not limited to heparin, warfarin, phenprocoumon, fondaparinux,lepirudin, bivalirudin, argatroban, danaparoid and drotrecogin alfa.

The term “anti-platelet agents” herein refers to compound or substancethat prevents platelet adhesion and/or aggregation. Examples ofanti-platelet agents include, but are not limited to prasugrel, aspirin,ticlopidine, clopidogrel, abciximab, eptifibatide and tirofiban.

The term “fibrinolytic agents” therefore refers to any compound orsubstance that lyses pathological thrombi. “Thrombolytic agents” areagents that are fibrinolytic, i.e., agents that convert plasminogen toplasmin, which lyses fibrin. Examples of fibrinolytic agents include butare not limited to streptokinase and tissue plasminogen activator(t-PA).

The term “stent” herein refers to expandable wire form or perforatedtube that is inserted into a natural conduit of the body, such as anartery, usually a coronary artery, to prevent or counteract adisease-induced localized flow constriction.

The term “optionally substituted” is intended to mean substituted withthe substituents defined or unsubstituted. For example, phenyloptionally substituted with one or more nitro means in some instances,the phenyl is substituted with one or more nitro groups and in otherinstances, the phenyl is unsubstituted.

The binding of LIBS-specific mAbs to αIIbβ3 may be measured by comparingthe binding of LIBS-specific mAbs to αIIbβ3 in the presence of testingcompound with the binding of LIBS-specific mAbs to αIIbβ3 in the absenceor presence of a control such as untreated platelets and/or other knownαIIbβ3 inhibitors that are known to cause β3 LIBS exposure, e.g.,tirofiban. For example, the test compound may bind to αIIb andoptionally increases binding of at least one αIIb LIBS-specific mAbrelative to binding to unactivated platelets without increasing thebinding of one or more β3 LIBS-specific mAbs relative to binding tounactivated platelets and/or produces less binding relative to bindingin the presence of an agent known to bind to and directly activateαIIbβ3 so as to expose β3 LIBS.

As used herein, the term “alkyl” or “alkyl chain” or “alkylene” refersto a linear or branched, saturated or unsaturated, aliphatichydrocarbon. Unless otherwise specified, alkyl refers to a hydrocarbonchain containing one to four carbon atoms. Examples of alkyl mayinclude, but are not limited to methyl, ethyl, tert-butyl and the likeas well as alkenyl or alkynyl substituents.

The term “C₃-C₁₀cycloalkyl” or “C₃₋₁₀cycloalkyl” refers to fully orpartially saturated, carbocyclic, non-aromatic hydrocarbon radicalshaving three to eight carbon atoms. Examples of C₃-C₁₀cycloalkyloptionally containing one or more heteroatoms selected from a groupconsisting of O or N include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl or cyclohexenyl,piperidinyl, piperazinyl, morpholinyl, imidazolinyl, pyrrolidinyl. Thesecycloalkyl systems may be attached via the heteroatom or any othercarbon on the system. C₃-C₁₀cycloalkyl may also refer to non-aromaticcyclic system fused to an aromatic cyclic system. An example of thisincludes tetrahydroquinolinyl.

The term “aryl” refers to any aromatic ring system. Aromatic compoundsinclude phenyl, naphthyl and their derivatives.

The term “heteroaryl” is intended to mean a stable 5- to 6-memberedmonocyclic or 7- to 14-membered bicyclic heterocyclic ring which issaturated partially unsaturated or unsaturated (aromatic), and whichconsists of carbon atoms and 1, 2, 3 or 4 heteroatoms independentlyselected from the group consisting of N, O and S and including anybicyclic group in which any of the above-defined heterocyclic rings isfused to another ring.

The term “acyl” is intended to encompass R—C(O)— wherein R is C₁₋₄alkylwherein said alkyl is optionally substituted with one or more halo,hydroxy, or C₁₋₄alkoxy.

The Compounds of the Invention may comprise one or more chiral carbonatoms. The compounds thus exist in individual isomeric, e.g.,enantiomeric or diasteriomeric form or as mixtures of individual forms,e.g., racemic/diastereomeric mixtures. Any isomer may be present inwhich the asymmetric center is in the (R)-, (S)-, or(R,S)-configuration. The invention is to be understood as embracing bothindividual optically active isomers as well as mixtures (e.g.,racemic/diasteromeric mixtures) thereof. Accordingly, the Compound ofthe Invention may be a racemic mixture or it may be predominantly, e.g.,in pure, or substantially pure, isomeric form, e.g., greater than 70%enantiomeric excess (“ee”), preferably greater than 80% ee, morepreferably greater than 90% ee, most preferably greater than 95% ee. Thepurification of said isomers and the separation of said isomericmixtures may be accomplished by standard techniques known in the art(e.g., column chromatography, preparative TLC, preparative HPLC,simulated moving bed and the like)

Compounds of the Invention may exist in free or salt form, e.g., as acidaddition salts (e.g., hydrochloric acid, toluene sulfonic acid, methanesulfonic acid, benzene sulfonic acid, trifluoroacetic acid, and thelike). In this specification unless otherwise indicated language such asCompounds of the Invention is to be understood as embracing thecompounds in any form, for example free or acid addition salt form, orwhere the compounds contain acidic substituents, in base addition saltform. The Compounds of the Invention are intended for use aspharmaceuticals, therefore pharmaceutically acceptable salts arepreferred. Salts which are unsuitable for pharmaceutical uses may beuseful, for example, for the isolation or purification of free Compoundsof the Invention or their pharmaceutically acceptable salts, aretherefore also included. In particular embodiment, the salt of thecompound of the invention is a trifluoroacetic acid addition salt.

Compounds of the Invention may in some cases also exist in prodrug form.A prodrug form is compound which converts in the body to a Compound ofthe Invention. For example, when the Compounds of the Invention containhydroxy or carboxy substituents, these substituents may formphysiologically hydrolysable and acceptable esters. As used herein,“physiologically hydrolysable and acceptable ester” means esters ofCompounds of the Invention which are hydrolysable under physiologicalconditions to yield acids (in the case of Compounds of the Inventionwhich have hydroxy substituents) or alcohols (in the case of Compoundsof the Invention which have carboxy substituents) which are themselvesphysiologically tolerable at doses to be administered. For example,wherein the compounds of the invention contains a hydroxy group (e.g.,Drug-OH), the prodrug (e.g., Drug-O—C(O)—CH₃) may hydrolyze underphysiological conditions to yield hydroxy (Drug-OH) on the one hand andacid, e.g., carboxylic acid on the other (e.g., CH₃COOH), which arethemselves physiologically tolerable at doses to be administered.Similarly, wherein the compounds of the invention contains a carboxylicacid group (e.g., Drug-C(O)OH), its prodrug (e.g., Drug-C(O)—O—CH₂CH₃)may hydrolyze under physiological conditions to yield the carboxylicacid (Drug-C(O)OH) on the one hand and alcohol, e.g., ethanol on theother (e.g., CH₃CH₂OH), which are themselves physiologically tolerableat doses to be administered. As will be appreciated the term thusembraces conventional pharmaceutical prodrug forms.

Compounds of the present invention may be administered orally orparenterally, including intravenous, intramuscular, intraperitoneal,subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical(including buccal and sublingual) administration. The compounds usefulin the invention may generally be provided in the form of tablets orcapsules, as a powder or granules, or as an aqueous solution orsuspension. Tablets for oral use may include the active ingredientsmixed with pharmaceutically acceptable excipients such as inertdiluents, disintegrating agents, binding agents, lubricating agents,sweetening agents, flavouring agents, colouring agents andpreservatives. Suitable inert diluents include sodium and calciumcarbonate, sodium and calcium phosphate, and lactose, while corn starchand alginic acid are suitable disintegrating agents. Binding agents mayinclude starch and gelatin, while the lubricating agent, if present,will generally be magnesium stearate, stearic acid or talc. If desired,the tablets may be coated with a material such as glyceryl monostearateor glyceryl distearate, to delay absorption in the gastrointestinaltract.

Dosages of the compounds of the invention will vary depending upon thecondition to be treated or prevented and on the identity of theinhibitor being used. Estimates of effective dosages and in vivohalf-lives for the individual compounds encompassed by the invention canbe made on the basis of in vivo testing using an animal model, such asthe mouse model described herein or an adaptation of such method tolarger mammals. Appropriate dosage may range from 0.01 mg to 5000 mg.For example, one appropriate dosage may be 0.01-30 mg/Kg, e.g., 26.5mg/Kg, e.g., 12 mg/Kg.

In addition to their administration singly, the compounds usefulaccording to the invention can be administered in combination or inconjunction with other known therapeutic agents useful for thromboticdisorders such as anticoagulants (e.g., heparin, warfarin,phenprocoumon, fondaparinux, lepirudin, bivalirudin, argatroban,danaparoid, drotrecogin alfa), fibrinolytic agents (e.g.: streptokinaseor tissue plasminogen activator (t-PA) or other anti-platelet agents(e.g., prasugrel, aspirin, ticlopidine, clopidogrel, abciximab,eptifibatide and tirofiban). In any event, the administering physiciancan adjust the amount and timing of drug administration on the basis ofresults observed using standard measures of platelet activity known inthe art or described herein.

EXAMPLES Example 1 Synthesis of Compounds of the Present Invention

The compounds described herein and their pharmaceutically acceptablesalts may be made using the methods as described and exemplified hereinand by methods similar thereto and by methods known in the chemical art.In the description of the synthetic methods described herein, it is tobe understood that all proposed reaction conditions, including choice ofsolvent, reaction atmosphere, reaction temperature, duration of theexperiment and workup procedures, are chosen to be the conditionsstandard for that reaction, which should be readily recognized by oneskilled in the art. Therefore, at times, reaction may require to be runat elevated temperature, for a longer or shorter period of time or inthe presence of an acid or base. It is understood by one skilled in theart of organic synthesis that functionality present on various portionsof the molecule must be compatible with the reagents and reactionsproposed. If not commercially available, starting materials for theseprocesses may be made by procedures, which are selected from thechemical art using techniques similar or analogous to the synthesis ofknown compounds. Significances of the substituents are as set forth inthe formulae hereinbefore defined unless otherwise specified. Allreferences cited herein are hereby incorporated in their entirety byreference.

All commercially available reagents and solvents are purchased and usedwithout further purification. All microwave reactions are carried out ina sealed microwave vial equipped with a magnetic stir bar and heated ina Biotage Initiator Microwave Synthesizer. All compounds for biologicaltesting are purified using a Waters semi-preparative HPLC equipped witha Phenomenex Luna® C18 reverse phase (5 micron, 30×75 mm) column havinga flow rate of 45 mL/min. The mobile phase is a mixture of acetonitrileand H₂O each containing 0.1% trifluoroacetic acid. During purification,a gradient of 30% to 80% acetonitrile over 8 minutes is used withfraction collection triggered by UV detection (220 nM). Pure fractionspassed through PL-HCO₃ MP SPE (Varian) to remove trifluoroacetic acidand concentrated under vacuum on a lyophilizer. ¹H spectra are recordedusing an Inova 400 (100) MHz spectrometer (Varian).

The Compounds of Formula P-I, any of 2.1-2.13, Formula Q-I, wherein R₂is H, Y is phenylene and R₁ is as defined in Formula P-I, any of2.1-2.13, or Formula Q-I, for example R₁ isN(H)—C(O)—C(R₁₁)(R₁₂)—N(R₁₄)(R₁₅), or —N(H)—C(O)-heterocycloalkyl,preferably N(H)—C(O)—CH₂NH₂ or —N(H)—C(O)—CH₂-piperidine, may beprepared by first reacting 5-(3-nitrophenyl)-1,3,4-thiadiazol-2-amine(Int-4) with methyl 3-chloro-3-oxopropanoate, which product is thenhalogenated, e.g., reacting with phosphorous oxychloride in the presenceof a base, e.g., diisopropyl ethyl amine or the like, optionally withheat, e.g., up to about 150° C., e.g., using microwave to produce7-chloro-2-(3-nitrophenyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one,(Int-5). Boc-protected piperazine is then reacted with Int-5 optionallyin the presence of heat, e.g., up to about 150° C., e.g., using amicrowave, to produce tert-butyl4-(2-(3-nitrophenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate,Int-6. The nitro group on Int-6 is then reduced to an amine, e.g., bycatalytic hydrogenation, e.g., using Raney Nickel and hydrazine toproduce tert-butyl4-(2-(3-aminophenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate,Int-7. Int-7 is then reacted with HOC(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄) in thepresence of a activating agent, e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Wherein R₁₃ and/or R₁₄ are H,HOC(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄) is preferably protected with a protectinggroup, e.g., a BOC protecting group, e.g. HOC(O)—C(R₁₁)(R₁₂)—N(R₁₃)— BOCor HOC(O)—C(R₁₁)(R₁₂)—N(R₁₄)-BOC. The resulting compound of Formula P-I,any of 2.1-2.13, or Formula Q-I is then deprotected, e.g., using acid,e.g., trifluoroacetic acid to provide the Compound of Formula P-I, anyof 2.1-2.13, or Formula Q-I wherein R₁ isN(H)—C(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄), e.g., N(H)—C(O)—CH₂NH₂. The synthesismay be summarized in the reaction scheme below:

Specifically, the synthesis for the Compound of Formula P-I, any of2.1-2.13, or Formula Q-I, wherein A is S, R₂ is H and R₁ is—N(H)—C(O)—CH₂NH₂ may be summarized in the reaction scheme below:

General Synthetic Procedures.

The synthesis methods described above and/or the following generalprocedures are used to synthesize compounds having different butanalogous structures. All final compounds differed only in thesubstitution off of the commercially available starting material.

TERMS AND ABBREVIATIONS

ACN=acetonitrile,

DCM=dichloromethane,

DMF=N,N-dimethylforamide,

DCM=dichloromethane

DIPEA=diisopropylethylamine

DMSO=dimethyl sulfoxide,

EDC=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

EtOAc=ethyl acetate,

h=hour(s),

HCl=hydrochloric acid

HPLC=high performance liquid chromatography,

K₂CO₃=potassium carbonate,

m=multiplet,

min.=minute(s)

MeOH=methanol,

MeCN=acetonitrile

MgSO₄=magnesium sulfate

NaHCO₃=sodium bicarbonate,

NMR=nuclear magnetic resonance,

p=pentet,

POCl₃=phosphorous oxychloride

rt=room temperature,

s=singlet,

t=triplet,

TFA=trifluoroacetic acid,

THF=tetrahydrofuran,

TLC=thin layer chromatography.

Example 12-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

Step 1: 7-chloro-2-ethyl-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

To a solution of 5-ethyl-1,3,4-thiadiazol-2-amines (5 g, 38.7 mmol, 1.0equiv) in 200 mL anhydrous acetonitrile is added to methyl3-chloro-3-oxopropionate (4.97 mL, 46.4 mmol, 1.2 equiv) and the mixtureis stirred for 2 h at room temperature. After consumption of thestarting material, phosphorous (V) oxychloride (40 mL, 429 mmol, 27equiv) is added along with N,N-diisopropylethylamine (6.76 mL, 38.7mmol, 1.0 equiv). The mixture is stirred at 150° C. for 25 min afterwhich it is concentrated in vacuo and taken up in chloroform, pouredover ice and washed with saturated NaHCO₃, water and brine. The organiclayer is dried (MgSO₄) and concentrated in vacuo to give a dark, redoil. The residue is purified by column chromatography (silica gel, 0-10%EtOAc/Dichloromethane) to give7-chloro-2-ethyl-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one (2.35 g,28.2% yield) as a tan solid.

Step 2: tert-butyl4-(2-ethyl-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate

To a solution of7-chloro-2-ethyl-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one (2.0 g,9.27 mmol, 1.0 equiv) in 50 mL of anhydrous acetonitrile in a microwavevial is added tert-butyl 1-piperazinecarboxylate (2.073 g, 11.13 mmol,1.2 equiv) followed by N,N-diisopropylethylamine (4.86 mL, 27.8 mmol,3.0 equiv). The mixture is heated in a microwave reactor for 25 min at150° C. Upon completion, the mixture is concentrated in vacuo to givecrude tert-butyl4-(2-ethyl-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylateas a yellow oil. This material is advanced directly to the next step.

Step 3:2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

To a solution of tert-butyl4-(2-ethyl-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate(3.39 g, 9.28 mmol, 1.0 equiv) in 40 mL of dichloromethane is addedtrifluoroacetic acid (10 ml, 130 mmol, 14 equiv). The mixture stirredfor 18 h, after which it is concentrated in vacuo and azeotroped withdichloroethane to give a yellow solid. The crude residue is dissolved inMeOH and purified by HPLC to give2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one(2.053 g, 83% yield) as an off-white solid. ¹H-NMR (400 MHz, DMSO-d₆) δppm 5.30 (s, 1H), 3.37-3.44 (m, 4H), 2.94 (q, J=7.56 Hz, 2H), 2.64-2.73(m, 4H), 1.25 (t, 3H); LCMS: (electrospray+ve), m/z 266.0 (MH)⁺; HPLC:t_(R)=2.71 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₁₁H₁₆N₅OS[M+H]⁺266.1069. found 266.1073.

Example 22-ethyl-7-(4-methylpiperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

This example is prepared by following the same procedures as describedin Example 1 above except substituting 1-methylpiperazine forBoc-piperazine. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 5.51 (s, 1H), 4.32(br. s., 2H), 3.39-3.75 (m, 4H), 3.01 (q, J=7.50 Hz, 2H), 2.83 (s, 3H),1.96-2.55 (m, 2H), 1.41 (t, 3H); LCMS: (electrospray+ve), m/z 280.1(MH)⁺; HPLC: t_(R)=2.69 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd forC₁₂H₁₈N₅OS [M+H]⁺280.1226. found 280.1224.

Example 37-(2,6-dimethylpiperazin-1-yl)-2-ethyl-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

This example is prepared by following the same procedures as describedin Example 1 above except substituting tert-butyl3,5-dimethylpiperazine-1-carboxylate for Boc-piperazine. ¹H NMR (400MHz, DMSO-d₆) δ ppm 5.32 (s, 1H), 4.03 (d, J=9.78 Hz, 2H), 2.92 (q,J=7.43 Hz, 2H), 2.53-2.66 (m, 2H), 2.18-2.30 (m, 2H), 1.23 (t, J=7.43Hz, 3H), 0.94 (d, 6H); LCMS: (electrospray+ve), m/z 294.2 (MH)⁺; HPLC:t_(R)=2.86 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₁₃H₂₀N₅OS[M+H]⁺294.1383. found 294.1382.

Example 42-ethyl-7-(4-ethylpiperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

This example is prepared by following the same procedures as describedin Example 1 above except substituting N-ethylpiperazine forBoc-piperazine. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 5.49 (s, 1H), 4.33(br. s., 2H), 3.42-3.75 (m, 4H), 3.12 (q, J=7.37 Hz, 2H), 3.01 (q,J=7.50 Hz, 2H), 2.76 (br. s., 2H), 1.32-1.45 (m, 6H); LCMS:(electrospray+ve), m/z 294.1 (MH)⁺; HPLC: t_(R)=2.76 min, UV₂₅₄=100%.HRMS (ESI): m/z calcd for C₁₃H₂₀N₅OS [M+H]⁺294.1383. found 294.1381.

Example 52-ethyl-6-methyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

This example is prepared by following the same procedures as describedin Example 1 above except substituting 3-ethoxy-2-methyl-3-oxopropanoicacid for 3-ethoxy-3-oxopropanoic acid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm3.09-3.16 (m, 4H), 2.94 (q, J=7.43 Hz, 2H), 2.66-2.75 (m, 4H), 1.88 (s,3H), 1.24 (t, J=7.63 Hz, 3H); LCMS: (electrospray+ve), m/z 280.1 (MH)⁺;HPLC: t_(R)=2.82 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₁₂H₁₈N₅OS[M+H]⁺280.1227. found 280.1230.

Example 62-ethyl-6-fluoro-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

This example is prepared by following the same procedures as describedin Example 1 above except substituting 3-ethoxy-2-fluoro-3-oxopropanoicacid for 3-ethoxy-3-oxopropanoic acid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm3.46-3.55 (m, 4H), 2.96 (q, J=7.43 Hz, 2H), 2.68-2.76 (m, 4H), 1.26 (t,3H); LCMS: (electrospray+ve), m/z 284.1 (MH)⁺; HPLC: t_(R)=2.59 min,UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₁₁H₁₅N₅OS [M+H]⁺284.0975. found284.0974.

Example 74-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)benzoicacid

This example is prepared by following the same procedures as describedin Example 1 above except substituting methyl4-(5-amino-1,3,4-thiadiazol-2-yl)benzoate for5-ethyl-1,3,4-thiadiazol-2-amine to obtain tert-butyl4-(2-(4-(methoxycarbonyl)phenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylateas a yellow oil. This oil (99 mg, 0.21 mmol, 1.0 equiv) is taken up in a1:1 THF/water mixture (3.0 mL), and to it is added lithium hydroxide (10mg, 0.420 mmol, 2.0 equiv). After stirring for 2 h, the mixture isfiltered and concentrated to give a yellow oil which is taken on crudeto the same deprotection step mentioned above to give4-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)benzoicacid as a tan solid 33 mg, 44%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.86(br. s., 1H), 8.07-8.14 (m, 2H), 7.99-8.06 (m, 2H), 5.59 (s, 1H), 4.06(br. s., 1H), 3.69-3.81 (m, 4H), 3.08-3.21 (m, 4H); LCMS:(electrospray+ve), m/z 358.1 (MH)⁺; HPLC: t_(R)=3.03 min, UV₂₅₄=100%.HRMS (ESI): m/z calcd for C₁₆H₁₆N₅O₃S [M+H]⁺358.0969. found 358.0969.

Example 85-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-carboxylicacid

This example is prepared by following the same procedures as describedin Example 1 above except substituting methyl5-amino-1,3,4-thiadiazole-2-carboxylate for methyl4-(5-amino-1,3,4-thiadiazol-2-yl)benzoate. ¹H NMR (400 MHz, DMSO-d₆) δppm 8.58 (br. s., 1H), 5.90 (s, 1H), 3.68-3.78 (m, 4H), 3.10-3.19 (m,4H); LCMS: (electrospray+ve), m/z 282.0 (MH)⁺; HPLC: t_(R)=0.55 min,UV₂₅₄=95%. HRMS (ESI): m/z calcd for C₁₀H₁₂N₅O₃S [M+H]⁺282.0655. found282.0658.

Example 97-(piperazin-1-yl)-2-(pyridin-4-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

This example is prepared by following the same procedures as describedin Example 1 above except substituting5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine for5-ethyl-1,3,4-thiadiazol-2-amine. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.80(dd, J=4.30, 1.57 Hz, 2H), 7.85 (dd, J=4.30, 1.57 Hz, 2H), 5.40 (s, 1H),3.42-3.51 (m, 4H), 2.66-2.75 (m, 4H); LCMS: (electrospray+ve), m/z 315.0(MH)⁺; HPLC: t_(R)=2.55 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd forC₁₄H₁₅N₆OS [M+H]⁺315.1023. found 315.1024.

Example 102-tert-butyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

This example is prepared by following the same procedures as describedin Example 1 above except substituting5-tert-butyl-1,3,4-thiadiazol-2-amine for5-ethyl-1,3,4-thiadiazol-2-amine. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 5.32(s, 1H), 3.40 (m, 4H), 2.67 (m, 4H), 1.35 (s, 9H); LCMS:(electrospray+ve), m/z 294.1 (MH)⁺; HPLC: t_(R)=2.52 min (4 min method),UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₁₃H₂₀N₅OS [M+H]⁺294.1383. found294.1382.

Example 112-(4-nitrophenyl)-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

This example is prepared by following the same procedures as describedin Example 1 above except substituting5-(4-nitrophenyl)-1,3,4-thiadiazol-2-amine for5-ethyl-1,3,4-thiadiazol-2-amine. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.39(d, J=9.00 Hz, 2H), 8.12-8.20 (m, 2H), 5.40 (s, 1H), 3.43-3.51 (m, 4H),2.64-2.75 (m, 4H); LCMS: (electrospray+ve), m/z 359.1 (MH)⁺; HPLC:t_(R)=2.60 min (4 min method), UV₂₅₄=100%. HRMS (ESI): m/z calcd forC₁₅H₁₅N₆O₃S [M+H]⁺359.0921. found 359.0919.

Example 122-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]oxadiazolo[3,2-a]pyrimidin-5-one

This example is prepared by following the same procedures as describedin Example 1 above except substituting 5-ethyl-1,3,4-oxadiazol-2-aminefor 5-ethyl-1,3,4-thiadiazol-2-amine. ¹H NMR (400 MHz, DMSO-d₆) δ ppm5.23 (s, 1H), 3.35-3.43 (m, 4H), 2.80 (q, J=7.56 Hz, 2H), 2.64-2.73 (m,4H), 1.23 (t, 3H); LCMS: (electrospray+ve), m/z 250.1 (MH)⁺; HPLC:t_(R)=2.41 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₁₁H₁₆N₅O₂[M+H]⁺250.1299. found 250.1302.

Example 132-phenyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

This example is prepared by following the same procedures as describedin Example 1 above except substituting 5-phenyl-1,3,4-thiadiazol-2-aminefor 5-ethyl-1,3,4-thiadiazol-2-amine. ¹H NMR (400 MHz, DMSO-d₆) δ ppm7.84-7.93 (m, 2H), 7.53-7.66 (m, 3H), 5.38 (s, 1H), 3.40-3.51 (m, 4H),2.65-2.77 (m, 4H); LCMS: (electrospray+ve), m/z 314.1 (MH)⁺; HPLC:t_(R)=3.37 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₁₅H₁₆N₅OS[M+H]⁺314.1070. found 314.1073.

Examples 14-15 are Prepared by Using General Procedure Described Below

In a microwave vial is added7-chloro-2-ethyl-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one (50 mg,0.232 mmol, 1.0 equiv) followed by the R-boronic acid (0.464 mmol, 2.0equiv), sodium carbonate (73.7 mg, 0.696 mmol, 3.0 equiv), andtetrakis(triphenylphosphine)palladium(0) (13.40 mg, 0.012 mmol, 0.05equiv). The solids are taken up in DMF (1.4 mL), Ethanol (0.700 mL), andWater (0.350 mL). The mixture is then heated in the microwave at 120° C.for 25 min. Upon completion, the mixture is taken up in ethyl acetateand saturated potassium hydrogen sulfate. The layers are separated andthe aqueous layer is extracted with ethyl acetate. The combined organicextracts are then washed with saturated sodium bicarbonate, water, andbrine. They are dried over magnesium sulfate and concentrated in vacuoto give a yellow oil which is purified by HPLC to give2-ethyl-7-aryl-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-ones (20-40%) astan solids.

Example 142-ethyl-7-(pyridin-3-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.21 (d, J=2.35 Hz, 1H), 8.61-8.68 (m,1H), 8.40 (ddd, J=8.02, 2.15, 1.96 Hz, 1H), 7.49 (dd, J=8.02, 4.89 Hz,1H), 7.07 (s, 1H), 3.04 (q, J=7.43 Hz, 2H), 1.30 (t, 3H); LCMS:(electrospray+ve), m/z 259.1 (MH)⁺; HPLC: t_(R)=2.98 min, UV₂₅₄=100%.HRMS (ESI): m/z calcd for C₁₂H₁₁N₄OS [M+H]⁺259.0648. found 259.0639.

Example 152-ethyl-7-(1,2,3,6-tetrahydropyridin-4-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

This example are prepared by following the same general procedure abovefor examples 14-15 to obtain tert-butyl4-(2-ethyl-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)-5,6-dihydropyridine-1(2H)-carboxylate,which is taken on to the same deprotection procedure as with2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-onedescribed in step 3 of Example 1. ¹H NMR (400 MHz, DMSO-d₆) δ ppm6.94-7.00 (m, 1H), 6.21 (s, 1H), 3.36 (d, J=3.13 Hz, 2H), 2.99 (q,J=7.43 Hz, 2H), 2.81 (t, J=5.48 Hz, 2H), 2.21 (d, 2H), 1.26 (t, J=7.43Hz, 3H); LCMS: (electrospray+ve), m/z 263.0 (MH)⁺; HPLC: t_(R)=2.32 min(4 min method), UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₁₂H₁₅N₄OS[M+H]⁺263.0960. found 263.0958.

Examples 16-26 are Prepared Using the General Procedures Below Step 1:5-(3-bromophenyl)-1,3,4-thiadiazol-2-amine

To a solution of thiosemicarbazide (0.493 g, 5.40 mmol, 1.0 equiv) in 6mL of water at 70° C. is added a solution of 3-bromobenzaldehyde (0.633ml, 5.40 mmol, 1.0 equiv) in 4.5 mL ethanol. After the precipitateformed, iron(III) chloride hexahydrate (2.92 g, 10.81 mmol, 2.0 equiv)in 6 mL water is added and the mixture is stirred for 2.5 h at 85° C.Upon completion, the mixture is allowed to cool to room temperature andthe resulting precipitate is collected by filtration and taken up in 12mL pyridine. Ice water is then added to the pyridine slurry. The mixtureis cooled then filtered to give5-(3-bromophenyl)-1,3,4-thiadiazol-2-amine (553 mg, 39.9% yield) as abrown solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.85-7.88 (m, 1H), 7.67 (d,J=8.22 Hz, 1H), 7.53-7.59 (m, 1H), 7.45 (s, 2H), 7.36 (t, J=8.02 Hz,1H);

Step 2

Boc-protected2-(3-bromophenyl)-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-onemay be prepared as similarly described in step 2 of Example 1. To asolution of tert-butyl4-(2-(3-bromophenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate(40 mg, 0.081 mmol, 1.0 equiv) in 1 mL DMF, 0.5 mL ethanol and 0.25 mLwater in a microwave vial is added the boronic acid (0.162 mmol, 2.0equiv), sodium carbonate (25.8 mg, 0.244 mmol, 3.0 equiv), andtetrakis(triphenylphosphine)palladium(0) (4.69 mg, 4.06 μmol, 0.05equiv). The mixture is heated in a microwave reactor for 30 min at 120°C. Upon completion the mixture is filtered through a thiol-SPE column(Stratospheres) and the resulting solution is concentrated in vacuo togive crude tert-butyl4-(5-oxo-2-(3-(aryl-4-yl)phenyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylateas a yellow oil. The residue is then taken on crude to the nextreaction, which followed the same deprotection procedure as described instep 3 of Example 1 to give7-(piperazin-1-yl)-2-(3-(aryl-4-yl)phenyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-ones(30-50%) as tan solids.

Example 167-(piperazin-1-yl)-2-(3-(pyridin-4-yl)phenyl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-one

Step 1: 5-(3-bromophenyl)-1,3,4-thiadiazol-2-amine

To a solution of thiosemicarbazide (0.493 g, 5.40 mmol, 1.0 equiv) in 6mL of water at 70° C. is added a solution of 3-bromobenzaldehyde (0.633ml, 5.40 mmol, 1.0 equiv) in 4.5 mL ethanol. After the precipitate isformed, iron(III) chloride hexahydrate (2.92 g, 10.81 mmol, 2.0 equiv)in 6 mL water is added and the mixture is stirred for 2.5 h at 85° C.Upon completion, the mixture is allowed to cool to room temperature andthe resulting precipitate is collected by filtration and taken up in 12mL pyridine. Ice water is then added to the pyridine slurry. The mixtureis cooled then filtered to give5-(3-bromophenyl)-1,3,4-thiadiazol-2-amine (553 mg, 39.9% yield) as abrown solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.85-7.88 (m, 1H), 7.67 (d,J=8.22 Hz, 1H), 7.53-7.59 (m, 1H), 7.45 (s, 2H), 7.36 (t, J=8.02 Hz,1H);

Step 2:7-(piperazin-1-yl)-2-(3-(pyridin-4-yl)phenyl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-one

Boc-protected2-(3-bromophenyl)-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-onemay be prepared as similarly described above (or in Steps 1 and 2 ofExample 1), except 5-(3-bromophenyl)-1,3,4-thiadiazol-2-amine is used asthe starting material in step 1.

To a solution of tert-butyl4-(2-(3-bromophenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate(40 mg, 0.081 mmol, 1.0 equiv) in 1 mL DMF, 0.5 mL ethanol and 0.25 mLwater in a microwave vial is added 4-pyridineboronic acid (19.97 mg,0.162 mmol, 2.0 equiv), sodium carbonate (25.8 mg, 0.244 mmol, 3.0equiv), and tetrakis(triphenylphosphine)palladium(0) (4.69 mg, 4.06μmol, 0.05 equiv). The mixture is heated in a microwave reactor for 30min at 120° C. Upon completion the mixture is filtered through athiol-SPE column (Stratospheres) and the resulting solution isconcentrated in vacuo to give crude tert-butyl4-(5-oxo-2-(3-(pyridin-4-yl)phenyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylateas a yellow oil. The residue is then taken on crude to the nextreaction, which follows the same deprotection procedure as described inStep 3 of Example 1 to give7-(piperazin-1-yl)-2-(3-(pyridin-4-yl)phenyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one(12 mg, 38%) as a tan solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.67 (dd,J=4.30, 1.57 Hz, 2H), 8.18 (s, 1H), 8.02 (dd, J=16.43, 8.22 Hz, 2H),7.70-7.82 (m, 3H), 5.40 (s, 1H), 3.42-3.51 (m, 4H), 2.68-2.75 (m, 4H);LCMS: (electrospray+ve), m/z 391.1 (MH)⁺; HPLC: t_(R)=2.82 min,UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₂₀H₁₉N₆OS [M+H]⁺391.1321. found391.1322.

Example 177-(piperazin-1-yl)-2-(3-(pyridin-3-yl)phenyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.96 (d, J=1.57 Hz, 1H), 8.61 (dd,J=4.70, 1.57 Hz, 1H), 8.14-8.20 (m, 1H), 8.12 (s, 1H), 7.93-8.01 (m,2H), 7.72 (t, J=7.83 Hz, 1H), 7.52 (dd, J=8.41, 5.28 Hz, 1H), 5.39 (s,1H), 3.41-3.50 (m, 4H), 2.66-2.75 (m, 4H); LCMS: (electrospray+ve), m/z391.1 (MH)⁺; HPLC: t_(R)=2.87 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd forC₂₀H₁₉N₆OS [M+H]⁺391.1334. found 391.1337.

Example 182-(3-(1H-pyrazol-4-yl)phenyl)-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

¹H NMR (400 MHz, DMSO-d₆) δ ppm 13.06 (br. s., 1H), 8.16 (s, 2H), 8.00(s, 1H), 7.85 (d, J=7.83 Hz, 1H), 7.70 (d, J=8.61 Hz, 1H), 7.55 (t,J=7.83 Hz, 1H), 5.39 (s, 1H), 3.44 (d, J=5.09 Hz, 4H), 2.65-2.74 (m,4H); LCMS: (electrospray+ve), m/z 380.1 (MH)⁺; HPLC: t_(R)=3.53 min,UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₁₈H₁₈N₇OS [M+H]⁺380.1288. found380.1290.

Example 192-(3-(6-fluoropyridin-3-yl)phenyl)-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.64 (d, J=2.74 Hz, 1H), 8.39 (td,J=8.22, 2.74 Hz, 1H), 8.11 (s, 1H), 7.97 (dd, J=8.22, 1.56 Hz, 2H), 7.72(t, J=8.02 Hz, 1H), 7.32 (dd, J=8.61, 2.35 Hz, 1H), 5.40 (s, 1H),3.42-3.50 (m, 4H), 2.67-2.75 (m, 4H); LCMS: (electrospray+ve), m/z 409.0(MH)⁺; HPLC: t_(R)=3.81 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd forC₂₀H₁₈FN₆OS [M+H]⁺409.1242. found 409.1233.

Example 202-(3-(2-fluoropyridin-4-yl)phenyl)-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.34 (d, J=5.28 Hz, 1H), 8.21 (t, J=1.66Hz, 1H), 8.00-8.12 (m, 2H), 7.71-7.81 (m, 2H), 7.65 (s, 1H), 5.39 (s,1H), 3.41-3.48 (m, 4H), 2.66-2.75 (m, 4H); LCMS: (electrospray+ve), m/z409.1 (MH)⁺; HPLC: t_(R)=3.82 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd forC₂₀H₁₈FN₆OS [M+H]⁺409.1244. found 409.1246.

Example 213′-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)biphenyl-4-carboxylicacid

¹H NMR (400 MHz, DMSO-d₆) δ ppm 13.04 (br. s., 1H), 8.76 (br. s., 1H),8.14-8.18 (m, 1H), 8.05 (d, J=8.61 Hz, 2H), 7.94-8.03 (m, 2H), 7.88 (d,J=8.61 Hz, 2H), 7.73 (t, J=8.02 Hz, 1H), 5.61 (s, 1H), 3.71-3.82 (m,4H), 3.16 (br. s., 4H); LCMS: (electrospray+ve), m/z 434.1 (MH)⁺; HPLC:t_(R)=3.79 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₂₂H₂₀N₅O₃S[M+H]⁺434.1281. found 434.1270.

Example 223′-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)biphenyl-3-carboxylicacid

¹H NMR (400 MHz, DMSO-d₆) δ ppm 13.15 (br. s., 1H), 8.76 (br. s., 1H),8.23 (t, J=1.57 Hz, 1H), 8.14 (t, J=1.66 Hz, 1H), 7.90-8.05 (m, 4H),7.72 (t, J=7.92 Hz, 1H), 7.64 (t, J=7.83 Hz, 1H), 5.61 (s, 1H),3.69-3.83 (m, 4H), 3.16 (br. s., 4H); LCMS: (electrospray+ve), m/z 434.1(MH)⁺; HPLC: t_(R)=3.94 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd forC₂₂H₂₀N₅O₃S [M+H]⁺434.1282. found 434.1281.

Example 233′-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)biphenyl-4-carboxamide

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.12 (t, J=1.66 Hz, 1H), 8.02-8.06 (m,1H), 7.89-8.01 (m, 3H), 7.79-7.86 (m, 2H), 7.70 (t, J=7.83 Hz, 1H), 7.39(br. s., 1H), 5.40 (s, 1H), 3.41-3.50 (m, 4H), 2.66-2.76 (m, 4H); LCMS:(electrospray+ve), m/z 433.1 (MH)⁺; HPLC: t_(R)=3.56 min, UV₂₅₄=100%.HRMS (ESI): m/z calcd for C₂₂H₂₀N₆O₂S [M+H]⁺433.1441. found 433.1447.

Example 244-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)thiophene-2-carboxylicacid

¹H NMR (400 MHz, DMSO-d₆) δ ppm 13.27 (br. s., 1H), 8.77 (br. s., 1H),8.37 (d, J=1.56 Hz, 1H), 8.24 (d, J=1.56 Hz, 1H), 8.15 (t, J=1.66 Hz,1H), 8.03 (ddd, J=8.12, 1.47, 1.17 Hz, 1H), 7.89 (ddd, J=8.22, 1.37,0.98 Hz, 1H), 7.65 (t, J=7.92 Hz, 1H), 5.60 (s, 1H), 3.70-3.83 (m, 4H),3.08-3.22 (m, 4H); LCMS: (electrospray+ve), m/z 440.1 (MH)⁺; HPLC:t_(R)=3.70 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₂₀H₁₈N₅O₃S₂[M+H]⁺440.0845. found 440.0852.

Example 252-(3-(2-aminopyrimidin-5-yl)phenyl)-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.64 (d, J=2.93 Hz, 2H), 8.00 (d, J=1.17Hz, 1H), 7.85 (dd, J=9.10, 8.12 Hz, 2H), 7.58-7.68 (m, 1H), 6.87 (s,2H), 5.39 (d, J=2.54 Hz, 1H), 3.45 (br. s., 4H), 2.71 (d, 4H); LCMS:(electrospray+ve), m/z 407.1 (MH)⁺; HPLC: t_(R)=3.10 min, UV₂₅₄=100%.HRMS (ESI): m/z calcd for C₁₉H₁₉N₈OS [M+H]⁺407.1389. found 407.1385.

Example 262-(4-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)-1H-pyrazol-1-yl)aceticacid

¹H NMR (400 MHz, DMSO-d₆) δ ppm 13.14 (br. s., 1H), 8.79 (br. s., 1H),8.33 (d, J=0.78 Hz, 1H), 7.96-8.05 (m, 2H), 7.81-7.89 (m, 1H), 7.70-7.77(m, 1H), 7.58 (t, J=8.02 Hz, 1H), 5.60 (s, 1H), 4.97 (s, 2H), 3.70-3.82(m, 4H), 3.16 (br. s., 4H); LCMS: (electrospray+ve), m/z 438.1 (MH)⁺;HPLC: t_(R)=3.34 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₂₀H₂₀N₇O₃S[M+H]⁺438.1343. found 438.1338.

Examples 27-40 are Prepared Using General Procedures Below Step 1:tert-butyl4-(2-(3-aminophenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate

tert-butyl4-(2-(3-nitrophenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate(220, 0.480 mmol, 1.0 equiv) is dissolved in 5 mL methanol. To thesolution is added palladium on carbon (51.1 mg). The reaction is stirredunder an atmosphere of hydrogen for 3 h. Upon completion, the mixture isfiltered through celite and concentrated in vacuo to give crudetert-butyl4-(2-(3-aminophenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylateas a yellow solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.37 (s, 1H),7.20-7.29 (m, 1H), 7.13 (d, J=7.83 Hz, 1H), 6.83 (dd, J=7.63, 1.76 Hz,1H), 5.46 (s, 1H), 3.87 (s, 2H), 3.45-3.65 (m, 8H), 1.48 (m, 9H);

Step 2

Crude tert-butyl4-(2-(3-aminophenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate(60 mg, 0.140 mmol, 1.0 equiv) prepared as described in step 1 above, istaken up in DMF (1.5 mL) and to it is added the carboxylic acid (0.210mmol, 1.5 equiv) then EDC (40.3 mg, 0.210 mmol, 1.5 equiv). The mixtureis stirred at r.t. for 24 h. Upon completion, the reaction is then takenup in water and ethyl acetate. The layers are separated and the aqueouslayer is extracted with ethyl acetate. The combined organic extracts arethen washed with water twice, then brine, dried over MgSO₄, andconcentrated in vacuo to give a yellow solid which is chromatographedwith 2-7% MeOH/DCM gradient to give N-acylated tert-butyl4-(2-(3-aminophenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylates.The residue is then taken on to the next reaction, which follows thesame deprotection procedure to give N-acylated2-(3-aminophenyl)-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-ones(40-60%) as tan solids after HPLC purification.

Example 272-amino-N-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)acetamide

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.31 (s, 1H), 7.86 (d, J=7.43 Hz, 1H),7.48-7.58 (m, 2H), 5.38 (s, 1H), 3.46 (br. s., 4H), 3.29 (s, 2H),2.67-2.76 (m, 4H); LCMS: (electrospray+ve), m/z 386.1 (MH)⁺; HPLC:t_(R)=2.70 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₁₇H₂₀N₇O₂S[M+H]⁺386.1394. found 386.1393.

In a particular instance, the compound of this example is prepared asdescribed or similarly described as follows: To5-(3-nitrophenyl)-1,3,4-thiadiazol-2-amine (2) (3.0 g, 13.50 mmol) inMeCN (120 ml) is added methyl 3-chloro-3-oxopropanoate (1.735 mL, 16.20mmol). The mixture is stirred at room temperature for 2 hours. Afterconsumption of the starting material, POCl₃ (60 mL, 644 mmol) is addedalong with DIPEA (2.358 mL, 13.50 mmol) in MeCN (10 mL). The mixture isheated in the microwave at 150° C. for 25 min, cooled, and concentratedin vacuo. The resulting slurry is taken up in chloroform, poured overice and washed with saturated NaHCO₃, water and brine, dried over MgSO₄,and concentrated in vacuo to give a dark red oil which is purified viacolumn chromatography on a 100 g snap column with 0-10% EtOAc/DCMgradient elution to give7-chloro-2-(3-nitrophenyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one(1.36 g, 4.41 mmol, 32.6% yield) as an orange solid.

To7-chloro-2-(3-nitrophenyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one(1.0 g, 3.24 mmol) in MeCN (30 ml) in a microwave vial is addedtert-butyl 1-piperazinecarboxylate (0.724 g, 3.89 mmol) followed byDIPEA (1.697 ml, 9.72 mmol). The mixture is heated in the microwave to150° C. for 25 min., cooled and concentrated in vacuo and purified viacolumn chromatography on a 50 g snap column with 0-10% MeOH/DCM gradientelution to give tert-butyl4-(2-(3-nitrophenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate(1.44 g, 3.14 mmol, 97% yield) as a tan solid.

To a solution of tert-butyl4-(2-(3-nitrophenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate(769 mg, 1.677 mmol) in EtOH (12 ml) at room temperature is added Raney®2400 nickel (98 mg, 1.677 mmol) followed by hydrazine (0.526 ml, 16.77mmol) dropwise and the mixture is stirred at room temperature for 4hours. The reaction is monitored by TLC and LMCMS and additional nickeland hydrazine is added until completion. The mixture is filtered overcelite and concentrated in vacuo to give crude tert-butyl4-(2-(3-aminophenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate(443 mg, 1.034 mmol, 61.6% yield) as a tan solid.

Crude tert-butyl4-(2-(3-aminophenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate(60 mg, 0.140 mmol) is taken up in DMF (1.5 mL) and to it is added theBoc-glycine (37 mg, 0.210 mmol) then EDC (40.3 mg, 0.210 mmol). Themixture is stirred at room temperature for 24 hours and the reactionmixture is then taken up in water and ethyl acetate. The layers areseparated and the aqueous layer is extracted with ethyl acetate. Thecombined organic extracts are then washed with water twice, then brine,dried over MgSO₄, and concentrated in vacuo to give a yellow solid whichis purified via column chromatography with 2-7% MeOH/DCM gradient togive tert-butyl4-(2-(3-(2-(tert-butoxycarbonylamino)acetamido)phenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate(crude material confirmed by LCMS analysis). The residue is then takenup in 2 mL of dichloromethane and trifluoroacetic acid (1 ml, 13.0 mmol)is added and the mixture is stirred for 18 hours. The solution is thenconcentrated in vacuo and washed with dichloroethane to give a crudeyellow solid that is dissolved in methanol and purified by preparativeHPLC to give2-amino-N-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)acetamide(40 mg, 49%) as a tan solid after HPLC purification.

Example 286-fluoro-N-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)nicotinamide

¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.72 (s, 1H), 8.84 (d, J=2.74 Hz, 1H),8.52 (td, J=8.12, 2.54 Hz, 1H), 8.40 (s, 1H), 8.04 (dt, J=7.43, 1.96 Hz,1H), 7.52-7.65 (m, 2H), 7.37 (dd, J=8.61, 2.35 Hz, 1H), 5.38 (s, 1H),3.40-3.49 (m, 4H), 2.65-2.76 (m, 4H); LCMS: (electrospray+ve), m/z 452.1(MH)⁺; HPLC: t_(R)=3.65 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd forC₁₈H₂₈FN₇O₂S [M+H]⁺452.1300. found 452.1305.

Example 293-amino-N-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)propanamide

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.26 (d, J=1.57 Hz, 1H), 7.77 (dt,J=6.41, 2.47 Hz, 1H), 7.46-7.54 (m, 2H), 5.37 (s, 1H), 3.40-3.49 (m,4H), 2.82 (t, J=6.46 Hz, 2H), 2.65-2.74 (m, 4H), 2.39 (t, 2H); LCMS:(electrospray+ve), m/z 400.1 (MH)⁺; HPLC: t_(R)=3.27 min, UV₂₅₄=100%.HRMS (ESI): m/z calcd for C₁₈H₂₂N₇O₂S [M+H]⁺400.1549. found 400.1546.

Example 30N-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)piperidine-4-carboxamide

¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.12 (s, 1H), 8.27 (s, 1H), 7.79 (d,J=7.04 Hz, 1H), 7.49 (s, 2H), 5.37 (s, 1H), 3.44 (br. s., 4H), 2.93 (br.s., 2H), 2.70 (br. s., 4H), 2.40 (m, 2H), 1.65 (br. s., 2H), 1.50 (br.s., 2H); LCMS: (electrospray+ve), m/z 440.2 (MH)⁺; HPLC: t_(R)=2.90 min,UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₂₁H₂₆N₇O₂S [M+H]⁺440.1862. found440.1860.

Example 31(R)-4-amino-5-oxo-5-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenylamino)pentanoicacid

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.26 (t, J=1.76 Hz, 1H), 7.83 (ddd,J=7.97, 1.81, 1.57 Hz, 1H), 7.52-7.67 (m, 2H), 5.57 (s, 1H), 3.95 (t,J=6.46 Hz, 1H), 3.70-3.81 (m, 4H), 3.09-3.18 (m, 4H), 2.32-2.42 (m, 2H),2.01-2.11 (m, 2H); LCMS: (electrospray+ve), m/z 458.2 (MH)⁺; HPLC:t_(R)=2.66 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₂₀H₂₄N₇O₄S[M+H]⁺458.1605. found 458.1602.

Example 32(S)-2-amino-N-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)-3-(thiazol-4-yl)propanamide

¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.79 (s, 1H), 9.10 (d, J=1.96 Hz, 1H),8.86 (br. s., 1H), 8.33 (br. s., 2H), 8.22 (t, J=1.76 Hz, 1H), 7.76(ddd, J=8.07, 1.71, 1.37 Hz, 1H), 7.53-7.63 (m, 1H), 7.51 (d, J=1.96 Hz,1H), 5.57 (s, 1H), 4.29 (br. s., 1H), 3.70-3.81 (m, 4H), 3.34-3.45 (m,2H), 3.10-3.20 (m, 4H); LCMS: (electrospray+ve), m/z 483.0 (MH)⁺; HPLC:t_(R)=3.00 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₂₁H₂₃N₈O₂S₂[M+H]⁺483.1379. found 483.1376.

Example 33(S)-2-amino-N-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)propanamide

¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.71 (s, 1H), 8.83 (br. s., 1H), 8.26(t, J=1.66 Hz, 1H), 8.14-8.23 (m, 2H), 7.82 (ddd, J=7.87, 1.81, 1.66 Hz,1H), 7.53-7.66 (m, 2H), 5.57 (s, 1H), 4.01 (br. s., 1H), 3.71-3.80 (m,4H), 3.16 (br. s., 4H), 1.46 (d, 3H); LCMS: (electrospray+ve), m/z 400.2(MH)⁺; HPLC: t_(R)=2.79 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd forC₁₈H₂₂N₇O₂S [M+H]⁺400.1550. found 400.1545.

Example 34(R)-2-amino-N-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)propanamide

¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.71 (s, 1H), 8.83 (br. s., 1H), 8.26(t, J=1.66 Hz, 1H), 8.13-8.23 (m, 2H), 7.82 (ddd, J=7.87, 1.81, 1.66 Hz,1H), 7.52-7.67 (m, 2H), 5.57 (s, 1H), 4.01 (br. s., 1H), 3.70-3.81 (m,4H), 3.16 (br. s., 4H), 1.46 (d, 3H); LCMS: (electrospray+ve), m/z 400.2(MH)⁺; HPLC: t_(R)=2.82 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd forC₁₈H₂₂N₇O₂S [M+H]⁺400.1550. found 400.1547.

Example 35(R)-2-amino-N-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)-3-(thiazol-4-yl)propanamide

¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.79 (s, 1H), 9.10 (d, J=1.96 Hz, 1H),8.86 (br. s., 1H), 8.33 (br. s., 2H), 8.22 (t, J=1.76 Hz, 1H), 7.76(ddd, J=8.07, 1.71, 1.37 Hz, 1H), 7.53-7.63 (m, 1H), 7.51 (d, J=1.96 Hz,1H), 5.57 (s, 1H), 4.29 (br. s., 1H), 3.70-3.81 (m, 4H), 3.34-3.45 (m,2H), 3.10-3.20 (m, 4H); LCMS: (electrospray+ve), m/z 483.2 (MH)⁺; HPLC:t_(R)=3.02 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₂₁H₂₃N₈O₂S₂[M+H]⁺483.1380. found 483.1381.

Example 36(R)-2-amino-5-oxo-5-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenylamino)pentanoicacid

¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.44 (s, 1H), 8.33 (s, 1H), 7.71-7.80(m, 1H), 7.45-7.56 (m, 2H), 5.57 (s, 1H), 3.74 (br. s., 4H), 3.14 (d,J=4.70 Hz, 4H), 2.55 (m, 1H), 2.50 (s, 2H), 2.03 (br. s., 2H); LCMS:(electrospray+ve), m/z 458.2 (MH)⁺; HPLC: t_(R)=2.82 min, UV₂₅₄=100%.HRMS (ESI): m/z calcd for C₂₀H₂₄N₇O₄S [M+H]⁺458.1603. found 458.1598.

Example 37(S)-4-amino-5-oxo-5-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenylamino)pentanoicacid

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.26 (t, J=1.76 Hz, 1H), 7.83 (ddd,J=7.97, 1.81, 1.57 Hz, 1H), 7.51-7.67 (m, 2H), 5.57 (s, 1H), 3.96 (t,J=6.46 Hz, 1H), 3.70-3.81 (m, 4H), 3.09-3.18 (m, 4H), 2.33-2.42 (m, 2H),2.01-2.11 (m, 2H); LCMS: (electrospray+ve), m/z 458.2 (MH)⁺; HPLC:t_(R)=2.78 min, UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₂₀H₂₄N₇O₄S[M+H]⁺458.1604. found 458.1620.

Example 38N-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)isoxazole-3-carboxamide

¹H NMR (400 MHz, DMSO-d₆) δ ppm 11.03 (s, 1H), 9.16 (d, J=1.76 Hz, 1H),8.72 (br. s., 1H), 8.48 (t, J=1.86 Hz, 1H), 8.02 (ddd, J=8.07, 2.10,1.17 Hz, 1H), 7.63-7.70 (m, 1H), 7.59 (t, J=8.02 Hz, 1H), 7.02 (d,J=1.76 Hz, 1H), 5.58 (s, 1H), 3.70-3.79 (m, 4H), 3.10-3.19 (m, 4H);LCMS: (electrospray+ve), m/z 424.1 (MH)⁺; HPLC: t_(R)=3.54 min,UV₂₅₄=100%. HRMS (ESI): m/z calcd for C₁₉H₁₈N₇O₃S [M+H]⁺424.1186. found424.1181.

Example 392-(methylamino)-N-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)acetamide

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.27 (t, J=1.66 Hz, 1H), 7.78 (ddd,J=7.68, 1.91, 1.76 Hz, 1H), 7.53-7.64 (m, 2H), 5.57 (s, 1H), 3.94 (s,2H), 3.70-3.79 (m, 4H), 3.10-3.20 (m, 4H), 2.63 (s, 3H); LCMS:(electrospray+ve), m/z 400.2 (MH)⁺; HPLC: t_(R)=2.74 min, UV₂₅₄=100%.HRMS (ESI): m/z calcd for C₁₈H₂₂N₇O₂S [M+H]⁺400.1557. found 400.1547.

Example 40N-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)isonicotinamide

¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.77 (s, 1H), 8.75-8.82 (m, 2H), 8.42(s, 1H), 8.02-8.10 (m, 1H), 7.86-7.91 (m, 2H), 7.54-7.67 (m, 2H), 5.38(s, 1H), 3.40-3.50 (m, 4H), 2.67-2.75 (m, 4H); LCMS: (electrospray+ve),m/z 434.1 (MH)⁺; HPLC: t_(R)=3.08 min, UV₂₅₄=100%. HRMS (ESI): m/z calcdfor C₂₁H₂₀N₇O₂S [M+H]⁺434.1399. found 434.1393.

Example 412-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenylamino)aceticacid

To a solution of tert-butyl4-(2-(3-aminophenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate(50 mg, 0.117 mmol, 1.0 equiv) in 1.0 mL tetrahydrofuran is addeddropwise a solution of tert-butyl bromoacetate (0.069 mL, 0.467 mmol,4.0 equiv) in 0.5 mL tetrahydrofuran and a solution ofN,N-diisopropylethylamine (0.082 mL, 0.467 mmol, 4.0 equiv) in 0.5 mLtetrahydrofuran. The mixture is heated in a microwave reactor for 30 minat 120° C. Upon completion, the mixture is taken up in water and EtOAc.The layers are separated and the aqueous layer is back-extracted withEtOAc. The combined organic extracts are washed with water and brine,and concentrated in vacuo to give crude tert-butyl4-(2-(3-(2-tert-butoxy-2-oxoethylamino)phenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylateas a yellow solid. The residue is then taken on crude to the nextreaction, which followed the same deprotection procedure as above togive2-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenylamino)aceticacid (18 mg, 40%) as a tan solid.

Example 422-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)benzamido)aceticacid

3-(7-(4-(tert-butoxycarbonyl)piperazin-1-yl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)benzoicacid (45 mg, 0.098 mmol, 1.0 equiv), is prepared using the proceduresimilarly described in Example 7, is dissolved in 1 mL indimethylformamide. To the solution is added glycine tert-butyl ester(0.016 ml, 0.118 mmol, 1.2 equiv), PyBOP (77 mg, 0.148 mmol, 1.5 equiv),then N,N-diisopropylamine (0.052 ml, 0.295 mmol, 3.0 equiv). The mixturestirred at room temperature for 18 h. Upon completion, the mixture istaken up in water and EtOAc. The layers are separated and the aqueouslayer is back-extracted with EtOAc. The combined organic extracts arethen washed with water twice, then brine, dried (MgSO₄) and concentratedin vacuo to give a yellow oil which is purified by chromatography(silica gel, 2-5% MeOH/dichloromethane) to give tert-butyl4-(2-(3-(2-tert-butoxy-2-oxoethylcarbamoyl)phenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate(41 mg, 73.0% yield) as a yellow oil. The residue is then taken on tothe next reaction, which follows the same deprotection procedure asabove to give2-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)benzamido)aceticacid (16 mg, 53.7% yield) as a white solid.

Example 437-(piperazin-1-yl)-2-(3-(pyridin-4-ylmethylamino)phenyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one

To a solution of tert-butyl4-(2-(3-aminophenyl)-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylate(60 mg, 0.140 mmol, 1.0 equiv) and 4-pyridinecarboxaldehyde (0.013 mL,0.140 mmol, 1.0 equiv) in 1 mL tetrahydrofuran in a microwave vial isadded dibutyltin dichloride (4.25 mg, 0.014 mmol, 0.1 equiv) andPhenylsilane (0.035 mL, 0.280 mmol, 2.0 equiv). The mixture is heated ina microwave reactor for 10 min at 100° C. Upon completion, the reactionmixture is filtered through a thiol-SPE column (Stratospheres) andconcentrated in vacuo to give crude tert-butyl4-(5-oxo-2-(3-(pyridin-4-ylmethylamino)phenyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-yl)piperazine-1-carboxylateas a yellow oil. The residue is then taken on crude to the nextreaction, which followed the same procedure as above to give7-(piperazin-1-yl)-2-(3-(pyridin-4-ylmethylamino)phenyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one(10 mg, 18%) as a white solid.

Example 44 Platelet Adhesion and/or Aggregation

The platelet adhesion assay is conducted by a modification of the assayas described in Blue et al., Blood 2008, 111, 1248, the contents ofwhich are incorporated by reference in their entirety. Thirtymicroliters of human fibrinogen (50 μg/mL) in Tris/saline (100 mM NaCl,50 mM Tris/HCl, pH 7.4; American Diagnostica, Stamford, Conn.) are addedto black, clear-bottom, untreated polystyrene, nonsterile 384-wellmicrotiter plate wells (Corning no. 3711; Acton, Mass.). Afterincubating at 22° C. for 1 hour, plates are washed 3 times withTris/saline, and wells are then blocked with HBMT (138 mM NaCl, 12 mMNaHCO₃, 10 mM HEPES, 2.7 mM KCl, 0.4 mM NaH₂PO₄, 0.1% glucose, 0.35%BSA, pH 7.4) for at least 1 hour. An additional wash is performed usingHBMT with 1 mM MgCl₂ and 2 mM CaCl₂. Calcein-labeled platelets (finalconcentration 1×10¹¹/L) are treated with selected Compounds of theInvention (final concentration of 100 μM, 30 μM, 10 μM or 1 μM) at 22°C. for 20 minutes. Thirty microliters of platelets are then added to thewells. After 1 hour of adhesion, wells are washed 3 times with HBMT-1 mMMgCl₂/2 mM CaCl₂ and the plates are read by a fluorescent microtiterplate reader (Envision; Perkin Elmer) to detect calcein fluorescence(490 nm excitation and 515 nm emission). Negative controls consist ofwells containing platelets without compounds of the invention. Positivecontrols are wells containing platelets and known inhibitors of αIIbβ3,including mAbs 7E3 and 10E5, and EDTA.

The platelet aggregation assay is conducted by modification of the assayas disclosed in Blue et al., Blood 2008, 111, 1248, the contents ofwhich are incorporated by reference in their entirety. Citratedplatelet-rich plasma (PRP), generated by the centrifugation of wholeblood at 650 g for 4 minutes at 22° C., is incubated in aggregometercuvettes with selected Compounds of the Invention (final concentrationof 100 μM, 30 μM, 10 μM or 1 μM) or controls for 15 minutes at 37° C.After 30 seconds in the aggregometer (Kowa AF-10E; Tokyo, Japan) at 37°C. with stirring, ADP (5-20 μM) is added to induce aggregation and thelight transmittance is measured for 8 minutes. The initial slopes ofaggregation in the presence of different concentrations of the Compoundbeing tested are used to generate an IC₅₀.

Platelet adhesion and/or aggregation studies of various Compounds of theInvention show that various exemplified compounds of the inventionexhibit an IC₅₀ value of less than 100 μM in a platelet aggregationstudy and/or inhibition of greater than 20%, in some instances, greaterthan 30% at a concentration of 100 μM in a platelet adhesion study.Selected results are shown in Table 1 below:

TABLE 1 P.Ad.A^(a) P.Ad.A^(a) P.Ag.A^(a) # R₁ % inhibition^(d) IC₅₀ ^(d)IC₅₀ ^(d)

18 19 20 21 22 3-(aminoacetamide) 3-((S)-2-aminopropanamide)3-((R)-2-aminopropanamide) 3-(2-(methylamino)acetamide)3-(piperidine-4-carboxamide) 92%^(b) 35%^(c) 69%^(c) 24%^(c) 64%^(c) 1.1 μM  8.2 μM >20 μM ND >20 μM 0.163 μM 0.916 μM  5.9 μM ND  8.6 μM^(a)P.Ad.A. = platelet adhesion assay. P.Ag.A. = platelet aggregationassay; ^(b)% inhibition at 30 μM; ^(c)% inhibition at 100 μM ^(d)%inhibition and IC₅₀ values were determined utilizing the plateletadhesion assay and platelet aggregation assay as described in Blue etal, Blood 2008, 111, 1248. ND = not determined.

In 5 separate experiments, Compound #18 described in Table 1 above (or2-amino-N-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)acetamideof Example 27 of the current invention) produces 83±7% (mean±SD)inhibition of platelet adhesion to fibrinogen at a concentration of 100μM, 80±13% inhibition at 30 μM, and 66±10% inhibition at 10 μM; thecomparable values for2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-one(a compound disclosed and claimed in U.S. Ser. No. 12/514,286) at thesame concentrations are 70±15%, 38±10%, and 22±8%. For furthercomparison, tirofiban produces 87±9% inhibition at 10 μM.

Compound #18 described in Table 1 above (or the compound of Example 27of the current invention) also inhibits ADP-induced platelet aggregationof citrated PRP with an IC₅₀ of 90±20 nM (n=4). By comparison,2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-oneof U.S. Ser. No. 12/514,286 has an IC₅₀ of 12 and 18 μM when testedtwice under the same conditions. When PPACK is used as the anticoagulantinstead of citrate, the IC₅₀ for Compound #18 (or Example 27 of thecurrent invention) is nearly 3-fold higher (220 nM; n=4). Therefore, thecompound of Example 27 of the current invention is more than onehundred-fold more potent in inhibiting platelet aggregation than2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-oneof U.S. Ser. No. 12/514,286.

Example 45

The effects of the Compounds of the Invention on platelet adhesionand/or aggregation may be studied as described in the examples below.

Human αIIb and Murine β3 (hαIIb/mβ3) Platelets.

The production of mice transgenic for the hαIIb gene locus may beproduced as described in Thorton et al., Blood (2005) 112:1180-1188, thecontents of which are incorporated by reference in their entirety. Thesemice may be crossed with mice homozygous for targeted disruption of themαIIb gene (Itga2b). The resulting mice are a mixture of C57B1/6 andSV129 backgrounds. The genotypes of mice containing the hαIIb transgeneand homozygous for the targeted disruption of mαIIb may be confirmed byPCR and direct assessment of surface expression of the receptors may beperformed on washed platelets prepared from platelet-rich plasma (PRP)as previously in Eslin et al., Blood (2004) 104:3173-3180; Basani etal., Blood (2009) 113:902-910. FITC-conjugated anti-human CD41 (HIPS;eBioscience, San Diego, Calif.) antibody is used to detect hαIIb andphycoerythrin-conjugated anti-mouse CD41 (MWReg30; BD Biosciences, SanJose, Calif.) antibody is used to detect mαIIb.

Murine αIIb and Human β3 (mαIIb/hβ) Platelets.

Human normal β3 cDNA may be excised from the pcDNA3 mammalian expressionvector (a kind gift of Dr. Peter Newman) and ligated into the mouse stemcell virus MigR1 vector containing an internal ribosome entry site(IRES)-green fluorescent protein (GFP) insertion prior to thepolyadenylation signal. Virus containing hβ3 cDNA is then generatedusing Ecopack 2-293 cells (ATCC, Manassas, Va.). Supernatant containingthe virus may be collected 72 h after transfection, passed through a0.45 μm filter, and stored at −70° C. until further use.

Fetal liver cell transplantation may be performed as per Zou et al.,Blood (2007) 109:3284-3290, with minor modifications. Fetal liver cellsare harvested from Itgb3−/− embryos on a mixed C57B1/6 and 129S6/SVEVbackground at E14.5-E16.5 and the cell suspension is enriched for CD34+hematopoietic progenitors using negative selection (EasySep, StemCellTechnologies, Vancouver, BC, Canada). Enriched cells may be thencultured overnight in media containing murine stem cell factor (100ng/ml), murine interleukin 6 (10 ng/ml) and murine interleukin 3 (20ng/ml; all from PeproTech, Rocky Hill, N.J.). Fetal cell cultures may beinfected at 0.5 transducing units (TDU) per cell on two consecutive dayswith MigR1-β3 viral supernatant in the presence of stem cell factor,interleukins 3 and 6 (concentrations as above), and hexadimethrinebromide (8 μg/ml; polybrene, Sigma). Infected cells (approximately1.5−2×10⁶/mouse; approximately 60% expressing GFP and hβ3) may then beinjected i.v. into a lethally irradiated WT (i.e., Itgβ3+/+) mouse (900rads of X-rays in two divided doses, three hours apart). Plateletstudies may be performed with blood obtained 5 weeks or more aftertransplantation.

Platelet Aggregation:

Blood may be drawn via cardiac puncture from anesthetized Sprague Dawleyrats (Taconic, Hudson, N.Y.), WT C57B1/6 mice (Jackson Laboratories, BarHarbor, Me.), and mice expressing hαIIb/mβ3 and diluted 1:1 with amixture of 4 parts 0.165 mM NaCl, 0.01 mM HEPES, pH 7.4 containing 2 mMCaCl₂ and 1 mM MgCl₂. Blood from consenting human volunteers is obtainedfrom a peripheral vein using a 19 gauge needle and anticoagulated with1:10 vol 3.8% sodium citrate. Platelet-rich plasma (PRP) may be isolatedby centrifugation at 22° C. at 350 g for 10 minutes (rats); 250 g for2.5 minutes (mice); or 650 g for 4 minutes (human). Mouse PRP samplesmay be adjusted to 400,000 platelets/μl with the buffer used fordilution and human PRP may be adjusted to 300,000 platelets/μl withplatelet-poor plasma. Samples of PRP may be either untreated orincubated for 5 minutes at 37° C. with the Compounds of the Invention(100 μM). Platelet aggregation may be induced by adding to PRP adenosinediphosphate (ADP) at 30 μM (rats and WT mice), 20 or 30 μM (hαIIb/mβ3mice), or 5 μM (humans), and light transmission may be measured overtime in an aggregometer (Kowa AG-10E, Tokyo, Japan) with stirring.Percent inhibition may be calculated by comparing the initial slope ofuntreated samples to the samples treated with the Compounds of theInvention.

Soluble Fibrinogen Binding:

Whole blood from WT mice, mice expressing hαIIb/mβ3, or mice expressingmαIIb/hβ3 on their platelets may be drawn from the retro-bulbar venousplexus into an equal volume of 200 μM PPACK (Calbiochem, Gibbstown,N.J.) in 165 mM NaCl. Samples may be diluted in HEPES-modified Tyrodebuffer [HBMT; 138 mM NaCl, 12 mM NaHCO₃, 10 mM HEPES(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), 2.7 mM KCl, 0.4mM NaH₂PO₄, 0.1% glucose, 0.35% BSA, pH 7.4] containing 50 μM PPACK, 2mM CaCl₂, 1 mM MgCl₂, and may be left untreated or incubated with theCompounds of the Invention (20 or 100 μM), RGDS (1 mM) or EDTA (10 mM).Alexa488-fibrinogen (WT and hαIIb/mβ3 mice; 200 μg/ml; Invitrogen,Carlsbad, Calif.) or Alexa647-fibrinogen (mαIIb/hβ3 mice) may be added,and samples may be activated with a PAR-4-activating peptide (AYPGKF,200 μM) and incubated at 37° C. for 30 minutes. Samples may then bediluted 1:10 in HBMT containing CaCl₂ and MgCl₂ as above and analyzed byflow cytometry. Fibrinogen binding may be calculated from the geometricmean fluorescence intensity of platelets (gated by forward and sidescatter for WT and hαIIb/mβ3 mice, and GFP intensity for mαIIb/hβ3mice). Fibrinogen binding to unactivated samples may be defined asbackground binding and PAR-4 peptide-induced fibrinogen binding tountreated samples may be used to establish maximal (100%) binding.Studies performed on WT platelets to assess whether the dilution stepperformed prior to analysis resulted in fibrinogen dissociationdemonstrated that samples analyzed immediately after dilution in bufferwithout or with Alexa488-fibrinogen (to maintain the same fibrinogenconcentration) may have similar net geometric mean fluorescentintensities (133 and 124 units, respectively). When analyzed 15 minafter dilution, the values may be identical (115 units), representing 86and 93% of the immediate values.

Ferric Chloride (FeCl₃) Carotid Artery Injury Model:

The protocol for FeCl₃-induced injury to the carotid artery may beadapted from Yorovoi et al., Blood (2003) 102-4006-4013 with minorchanges. Four C57BL/6 WT mice, 6 WT mice on a mixed C57BL/6 and SV129background, and 16 hαIIb/mβ3 mice may be anesthetized by intraperitoneal(i.p.) injection of pentobarbital (80 mg/kg Nembutol; OvationPharmaceuticals, Deerfield, Ill.). After 10 min, mice may be injectedi.p. with 10 mM one of the Compounds of the Invention (26.5 mg/kg) (n=8)or the vehicle [1% (n=2) or 10% (n=6) dimethyl sulfoxide (DMSO) in 0.165M NaCl]. The carotid artery may then be isolated by blunt dissection anda Doppler flow probe (Model 0.5VB; Transonic Systems, Ithaca, N.Y.) maybe positioned around the vessel. Approximately 25 min after the compoundor vehicle control is administered, a 1×2 mm² piece of filter paper (#1;Whatman International, Maidstone, Kent, UK) soaked in 20% FeCl₃ may beplaced on the artery for 3 min and then removed. The area may then beflushed with distilled water and blood flow through the artery ismonitored for 30 minutes. Arterial flow rate data may be analyzed asboth “percent reduction in flow” (calculated as the area above the lineof the plot of observed flow rate versus observation time, divided bythe product of the initial flow rate and the total observation time) and“time to occlusion” (defined as the time from the application of theFeCl₃-soaked filter paper until arterial blood flow became undetectablefor at least 10 min).

Carotid arteries from one hαIIb/mβ3 mouse treated with 10% DMSO and onetreated with the Compounds of the Invention may be fixed informaldehyde, cross-sectioned, and stained with hematoxylin and eosin.The sections may be visualized with a 20× objective using an OlympusBX60 microscope (Melville, N.Y.), photographed with a Nikon D5-5Mcamera, and captured in Adobe Photoshop 6.0.

Laser Microvascular Injury and Intravital Microscopy:

The protocols for laser microvascular injury in blood vessels in thecremaster muscle and intravital microscopic evaluation of subsequentthrombus formation are described in Neyman et al., Blood (2008)112-1104-1108. Briefly, male mice expressing hαIIb/β3 (three in eachgroup) that are anesthetized with pentobarbitol (11 mg/kg; AbbottLaboratories, North Chicago, Ill.) may have their cremaster arterioles(20 to 40 μm) studied using an Olympus BX61WI microscope (Olympus,Tokyo, Japan or Center Valley, Pa.) with a 40×/0.8 numeric aperture (NA)water-immersion objective lens. Arteriole laser injuries may be doneusing an SRS NL100 Nitrogen Laser system (Photonic Instruments, StCharles, Ill.) at 65% energy level. Visual confirmation of theextravasation of small amounts of blood cells may be made for eachstudied blood vessel as an assurance that a consistent injury had beenproduced. After the surgery to expose the blood vessels is performed,animals may be injected i.p. with 10 mM the Compounds of the Inventionor 10% DMSO. Three arterioles may be injured in each mouse. Injuries maybe initiated 25-35 min after injection of the Compounds of the Inventionor DMSO, and 5 min after the i.v. injection of labeled antibodies thatreact with murine platelet GPIbβ (DyLight⁴⁸⁸; Emfret Analytics,Eibelstadt, Germany) and fibrin (Alexa⁶⁴⁷) into the cannulated jugularvein. Data may be collected over 2.5 min at 5 frames per second (750frames per study) and then averaged at each time point.

Results

Platelet Aggregation:

It will be observed that the Compounds of the Invention at 100 μM willdramatically inhibits aggregation of platelets from humans, and miceexpressing the hybrid hαIIb/mβ3 receptors, but not WT mice or rats. In aparticular experiment, it is actually observed that at doses that nearlycompletely inhibited platelet aggregation, the compound of Example 27 ofthe current invention at 1 μM does not inhibit either mouse or ratplatelet aggregation induced by ADP. In sharp contrast, this compoundessentially completely inhibits the aggregation of platelets from amouse expressing human αIIb and β3 (hαIIb/mβ3). Therefore, it is shownthat the compound of Example 27 of the current invention is selectivefor αIIbβ3 compared to αVβ3 and is further selective for human WINDcompared to mouse or rat αIIbβ3.

Soluble Fibrinogen Binding.

Activated platelets from WT mice will bind fibrinogen, and this bindingis expected to only minimally be inhibited by various Compounds of theInvention at 20 or 100 μM. Fibrinogen may also bind to activatedplatelets from mice expressing mαIIb/hβ3 and these receptors are alsonot expected to be inhibited by various Compounds of the Invention. Insharp contrast, binding of fibrinogen to activated platelets fromhαIIb/mβ3 mice is expected to be inhibited at a concentration of 20 μMand at 100 μM by one of the Compounds of the Invention. For comparisonEDTA will inhibit the binding of fibrinogen to WT mouse platelets andhαIIb/mβ3 mouse platelets. Thus, the effects of the Compounds of theInvention on fibrinogen binding to WT and hαIIb/mβ3 platelets willparallel its effects on platelet aggregation.

The Compounds of the Invention Will Protect hαIIb/mβ3 Mice, but Not WTMice, Against Occlusive Carotid Artery Thrombi.

The platelet counts and WIND expression levels of mice receiving theDMSO vehicle control solutions or the Compounds of the Invention willnot differ significantly. All 8 hαIIb/mβ3 mice treated with DMSO (1 or10%) will have reductions in carotid artery blood flow after FeCl₃injury. The hαIIb/mβ3 mice may be treated with the Compounds of theInvention. These mice will exhibit much less reduction in blood flow andwill not develop an occlusive thrombus.

Cross sections of the hαIIb/mβ3 mouse carotid artery after FeCl₃treatment will reveal nearly complete packing of the lumen withplatelet-rich thrombus. The deposits of FeCl₃ will be visible as goldengranules on the luminal side of the blood vessel. The lumen of the mousecarotid artery treated with the Compounds of the Invention will containregions of loosely packed erythrocytes, which will demonstrate thepatency of the artery. Golden FeCl₃ granules will also be visible on theluminal side of the blood vessel.

The Compounds of the Invention Will Decrease Thrombus Formation inResponse to Microvascular Laser Injury.

Cremaster arteriole injury studies will permit in situ visualization ofthrombus development in real time. There will be no difference inthrombus formation in mice that are either untreated or pre-injectedwith the carrier, 10% DMSO, prior to injury (comparison not shown). Bothgroups will demonstrate rapid platelet adhesion followed by progressiveincorporation of platelets into the thrombi over the first ˜20 sec. Inaddition, after a delay of ˜35 sec, both groups will demonstrateprogressive incorporation of fibrin into the thrombi. The animalspre-injected with the Compounds of the Invention prior to injury willnot develop platelet accumulation or fibrin deposition over a comparabletime period.

Example 46 Platelet Adhesion/Aggregation to Collagen; a/VP-Mediated CellAdhesion to Vitronectin; and αIIbβ3-Mediated Cell Adhesion to Fibrinogen

Washed platelets are prepared as per the primary screening assay,suspended in HBMT containing 1 mM MgCl₂ (platelet adhesion to collagen)or 2 mM CaCl₂/1 mM MgCl₂ (platelet adhesion to fibrinogen) and adjustedto a count of 250×10⁹/L. HEK293 cells stably expressing normal humanαVβ3 or normal human αIIbβ3 are suspended in HBMT containing either 1 mMMgCl₂ (HEK293 cells expressing αVβ3 for adhesion to vitronectin) or 2 mMCaCl₂/1 mM MgCl₂ (HEK293 cells expressing αIIbβ3 for adhesion tofibrinogen); the cell counts are adjusted to 10⁶/mL. Polystyrene 96-wellmicrotiter plates (Nunc) are coated with either fibrinogen (50 μg/mL),vitronectin (5 μg/mL), or collagen (33 μg/mL, rat tail type 1; BectonDickinson) for 1 hour, and blocked with HBMT for at least 1 hour.Platelets and cells are treated with the compound of Example 27 of thecurrent invention or2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-oneof U.S. Ser. No. 12/514,286 in the presence or absence of anti-α2β1 mAb6F1, anti-αIIbβ3 mAb 10E5, anti-αIIbβ3 mAb 10E5+αVβ3 mAb 7E3,αVβ3-specific mAb LM609, or anti-αVβ3+αIIbβ3 mAb 7E3, for 15 minutes at37° C. before being added to the microtiter wells. After adhering for 1hour at either 22° C. (platelets) or 37° C. (cells), nonadherentplatelets or cells are removed by washing 3 times with HBMT containingthe same ion composition as the buffer used for adhesion. Adherentplatelets or cells are quantified by their endogenous acid phosphataseactivity on p-nitrophenyl phosphate (pNPP) as previously described inLaw D A, Nannizzi-Alaimo L, Ministri K, et al., Blood (1999)93:2645-2652, the contents of which are incorporated by reference intheir entirety (1 mg/mL in 0.1 M sodium citrate, 0.1% Triton X-100, pH5.4). In other experiments, 8-chambered glass coverslips (Nunc) arecoated with collagen (33 μg/mL) for 1 hour at 22° C. Washed plateletsare allowed to adhere for 1 hour at 22° C. and the coverslips arestained with the Alexa-488-conjugated β3-specific mAb 7H2. Adherentplatelets are imaged using a Zeiss LSM-510 confocal system with Axiovert200 microscope (Carl Zeiss, Heidelberg, Germany) using a Plan-Apochromat100/1.4 NA oil DIC objective.

This experiment shows that anti-α2β1 mAb 6F1 produced 95% inhibition ofplatelet adhesion/aggregation to collagen, whereas the anti-αIIbβ3 mAb10E5 and the anti-αIIbβ3+αVβ3 mAb 7E3 produce ˜30% inhibition. Thecompound of Example 27 of the current invention at 1-100 μM alsoinhibits adhesion/aggregation by about ˜30%, and combining the compoundof Example 27 of the current invention with the anti-αIIbβ3 antibody10E5 does not further inhibit adhesion/aggregation. Microscopic analysisindicates that the compound of Example 27 of the current invention doesnot decrease platelet adhesion to collagen, but rather decreases therecruitment of additional platelets to the adherent platelets.

Further, the αVβ3-specific mAb LM609 inhibits adhesion of HEK293 cellsexpressing αVβ3 to vitronectin by 74±27% (n=4) at 20 μg/ml and theanti-αVβ3+αIIbβ3 mAb 7E3 inhibits adhesion by 80±12% (n=4) at 40 μg/ml.In sharp contrast,2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-oneof U.S. Ser. No. 12/514,286 at 100 μM produced only 5±7% (n=4)inhibition and2-amino-N-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)acetamideat 10 μM produced only 6±15% (n=4). These data are both similar to the2±9% (n=3) inhibition produced by the αIIbβ3-specific mAb 10E5.

αVβ3-specific mAb LM609 does not inhibit the adhesion of HEK293 cellsexpressing αIIbβ3 to fibrinogen, whereas 10E5 produces 79±10%inhibition, 7E3 produces 87±9% inhibition,2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-oneof U.S. Ser. No. 12/514,286 produced 55±5% inhibition, and the compoundof Example 27 of the current invention of the current invention produces65±5% inhibition (all n=4) at the same concentrations indicated for theαVβ3 experiments.

Example 47 Induction of Ligand-Induced Binding Site (LIBS) Epitopes

Washed platelets are prepared in HBMT containing 2 mM CaCl₂/1 mM MgCl₂.Platelet count is adjusted to 250×10⁹/L and the compound of Example 27of the current invention (100 μM),2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-one(a compound disclosed and claimed in U.S. Ser. No. 12/514,286),eptifibatide (10 μM), EDTA (10 mM), or mAb 10E5 (20 μg/mL) is added tosamples (30 μL) and incubated for 15 minutes at 37° C. Fluorescentlylabeled monoclonal antibodies (PMI-1, LIBS-1, AP5, or 10E5; Alexa-488conjugated; Invitrogen) are then added (20 μL, final concentration: 5μg/mL) and incubated for 30 minutes at 22° C. in the dark, after whichsamples are diluted 1:10 in HBMT with CaCl₂/MgCl₂ for analysis by flowcytometry (FACSCalibur; Becton Dickinson, Franklin Lakes, N.J.).Antibody binding is reported as the geometric mean fluorescenceintensity; nonspecific binding is determined by adding a 50-fold excessof unlabeled antibody before adding the labeled antibody. The netnormalized fluorescence intensity in the presence of eptifibatide (10μM, 30 min incubation at 22° C.) is assigned the value of 100%

This experiment shows that untreated platelets bind 7±3% (n=5) of theamount of AP5 in the presence of eptifibatide. In the presence of2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-oneof U.S. Ser. No. 12/514,286, platelets bind 10±4% of the amount in thepresence of eptifibatide. In the presence of the compound of Example 27of the current invention, platelets bind 18±5% of the amount in thepresence of eptifibatide. The comparable data for LIBS1 binding after 30min are 22±3% (n=5) for untreated platelets, 18±2% for platelets in thepresence of2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-one(a compound disclosed and claimed in U.S. Ser. No. 12/514,286), and21±3% for platelets in the presence of the compound of Example 27 of thecurrent invention. Untreated platelets bind 46±5% (n=4) of the amount ofPMI-1 that platelets treated with eptifibatide bound; both2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-oneof U.S. Ser. No. 12/514,286 and the compound of Example 27 of thecurrent invention increase PMI-1 binding to similar extents (73±9% and82±13, respectively).

In summary, Examples 46-47 show that the compound of Example 27 of thecurrent invention as well as2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-oneof U.S. Ser. No. 12/514,286 produce much less exposure than doeseptifibatide of the β3 PSI epitope recognized by mAb AP5. The compoundof Example 27 of the current invention does, however, producesignificantly more exposure than does2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-oneof U.S. Ser. No. 12/514,286 (18 and 10% of the eptifibatide value,respectively), with untreated platelets binding ˜7% of the eptifibatidevalue. Both2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-oneof U.S. Ser. No. 12/514,286 and the compound of Example 27 of thecurrent invention induce exposure of the αIIb leg region epitoperecognized by PMI-1, although they are less effective than eptifibatide.Thus, both compounds clearly affect the conformation of the αIIbsubunit, but have much less effect than eptifibatide, a prototypicRGD-mimetic antagonist, in inducing a conformational change in the β3subunit.

Example 48 The Effect of the Compound of the Invention on Induction ofExtension of Purified αIIbβ3 as Judged by Electron Microscopy

Purification of Integrin αIIβ3:

αIIbβ3 is purified from outdated single donor platelet concentratesobtained from the New York Blood Center by washing platelets in thepresence of PGE₁; removing contaminating blood cells by centrifugation;lysing the resuspended platelets at 4° C. in 5% (w/v)n-octyl-β-D-glucoside (OG) in 150 mM NaCl, 20 mM HEPES, 1 mM CaCl₂, 1 mMMgCl₂, 10 μM leupeptin, pH 7.4; performing Con A affinity chromatography[binding buffer: 150 mM NaCl, 1% (w/v) OG, 20 mM HEPES, 1 mM CaCl₂, 1 mMMgCl₂, pH 7.4; washing buffer: binding buffer+20 mM α-methyl glucoside;elution buffer: binding buffer+1M α-methyl glucoside]; performingheparin affinity chromatography; applying the flow through fraction toQ-Sepharose [binding buffer: 75 mM NaCl, 1% (w/v) OG, 10 mM HEPES, 1 mMCaCl₂, 1 mM MgCl₂, pH 7.4; washing buffer: binding buffer+200 mM NaCl;elution buffer: binding buffer+400 mM NaCl]; and performing gel sizeexclusion chromatography on Sephacryl S300 HR [running buffer: 150 mMNaCl, 1% (w/v) OG, 10 mM HEPES, 1 mM CaCl₂, 1 mM MgCl₂, pH 7.4].

Preparation of αIIbβ3-Containing Nanodiscs:

αIIbβ3-containing nanodiscs are prepared by a modification of thetechniques described by Ritchie et al., Babyurt and Sligar, and Ye etal²⁻⁴, the contents of which are incorporated by reference in theirentirety. In brief, the His-tagged membrane scaffold protein is preparedas a recombinant protein in E. coli and purified by Nickel affinitychromatography and anion exchange chromatography. Final assemblyconsists of solubilizing an equimolar mixture of1,2-dimyristoyl-sn-glycero-3-phosphocholine and1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) in octylglucosideand cholate and then adding the purified αIIbβ3. The detergents areremoved with macroporous polymeric beads (Bio-Bead SM-2) and then theαIIbβ3 nanodiscs are separated from the empty nanodiscs by gelfiltration.

Negative Staining Electron Microscopy and Evaluation of αIIbβ3 NanodiscParticle Size:

αIIbβ3 nanodiscs are treated with eptifibatide, tirofiban,2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-oneof U.S. Ser. No. 12/514,286, or the compound of Example 27 of thecurrent invention for 1 hour at room temperature at concentrations up to100 μM for2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-one(a compound claimed in U.S. Ser. No. 12/514,286) and at concentrationsup to 10 μM for2-amino-N-(3-(5-oxo-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-2-yl)phenyl)acetamideof Example 27. Samples are loaded onto carbon-coated copper grids thatare glow-discharged by a carbon coating unit (Edwards; Crowley, UK), andthen stained with 2% uranyl acetate, followed by drying. Imaging ofαIIbβ3 nanodiscs is performed using a JOEL JEM 100CX transmissionelectron microscope (Jeol Ltd; Tokyo, Japan) at 80 kV and magnificationsof 33,000× and 50,000×. Individual nanodiscs containing αIIbβ3 aremanually selected for analysis using Image J (NIH, Bethesda, Md.). Thedistance from the bottom of the nanodisc to the height of the αIIbβ3complex (nanodisc-integrin length; NIL) is measured as an indicator ofintegrin extension. The frequency distribution of NIL values is thenanalyzed for untreated αIIbβ3 nanodiscs and αIIbβ3 nanodiscs in thepresence of different compounds.

This experiment shows that in the absence of compound, αIIbβ3 primarilyadopts a compact conformation adjacent to the nanodisc, givingnanodisc-integrin length (NIL) values primarily between 11 and 17 nm.Occasional nanodiscs, however, contain αIIbβ3 molecules that areextended, giving NIL values between 18 and 23. As a result, the NILfrequency distribution shows a bimodal pattern, with a markedpredominance of αIIbβ3 nanodiscs in the range of 11 to 17 nm, and asmall subpopulation in the range 18 to 23 nm. Both eptifibatide andtirofiban shift the distribution in a dose-dependent manner such that atthe highest doses the majority of αIIbβ3 nanodiscs has NIL values in the18 to 23 nm range (p<0.001 and p<0.001, respectively). In sharpcontrast, neither2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-oneof U.S. Ser. No. 12/514,286 at concentrations up to 100 μM nor thecompound of Example 27 of the current invention at concentrations up to10 μM produce a significant shift in NIL values (p=0.23 and p=0.37,respectively). In other words, the compound of Example 27 of the currentinvention and2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-oneof U.S. Ser. No. 12/514,286 at these concentrations do not inducereceptor extension as judged by electron microscopy.

Example 49 The Effect of Mg²⁺ Concentration on Platelet Adhesion toFibrinogen in the Presence of the Compound of the Invention

Washed platelets in buffers containing 1 mM Ca²⁺ plus 1 mM, 20 mM, or 50mM Mg²⁺ are added to wells pre-coated with fibrinogen (50 μg/ml) for 60min at 22° C. After washing, adherent platelets are detected by acidphosphatase activity. Results are expressed as mean±SD (n=3 for thecompound of Example 27 of the current invention and2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-one(a compound disclosed and claimed in U.S. Ser. No. 12/514,286); n=2 fortirofiban).

In three separate experiments, the IC₅₀ for the compound of Example 27of the current invention increases from 0.29±0.1 μM (mean±SD) at 1 mMMg²⁺ to 0.91±0.21 μM at 20 mM Mg²⁺ (p<0.01), and to 1.3±0.35 μM at 50 mMMg²⁺ (p<0.01). Thus, in going from 1 to 50 mM Mg²⁺, there is an˜4.5-fold increase in IC₅₀, corresponding to nearly an ˜80% decrease inaffinity. Neither2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-oneof U.S. Ser. No. 12/514,286 nor tirofiban shows a comparable increase inIC₅₀ at higher Mg²⁺ concentrations.

Example 50 Effect of Mg²⁺ on Platelet Aggregation Induced by ADP in thePresence of Test Compound

Washed platelets are resuspended in buffer containing fibrinogen (200μg/ml) and 1 mM Mg²⁺ in combination with either 1 mM or 20 mM Mg²⁺.Aggregation is induced by adding a thrombin receptor activating peptide(SFLLRN) at 10 μM.

The inhibitory effect of the compound of Example 27 of the currentinvention (1 μM) on platelet aggregation induced by a thrombin receptoractivating peptide (SFLLRN) at different Mg²⁺ concentrations is clearlyattenuated at 20 mM Mg²⁺, whereas the effect on2-ethyl-7-(piperazin-1-yl)-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidin-5-oneof U.S. Ser. No. 12/514,286 (100 μM) is much less evident. Thisexperiment shows that the compound of Example 27 binds by a novelmechanism leading to loss of the MIDAS metal ion while producing onlyminor changes in the conformation of αIIbβ3.

What is claimed is:
 1. A compound of Formula P-I:

wherein: i) A is S, N(H), CH₂, or O; ii) R₁ is: phenyl optionallysubstituted with one or more nitro, —C(O)N(R₅)(R₆) and/or —N(R₅)(R₆) andY is a —C₁-C₄alkylene (e.g., methylene) or arylene; phenyl substitutedwith one or more nitro and/or —N(R₅)(R₆) and Y is a single bond; phenylsubstituted with —C(O)OR₃ and Y is a —C₁-C₄alkylene or arylene;heteroaryl wherein said heteroaryl group is optionally substituted withone or more —C₁-C₄alkyl and Y is a single bond, or —C₁-C₄alkylene;heteroaryl wherein said heteroaryl group is substituted with halo,C(O)OH, —CH₂C(O)OH, or —NH₂, and Y is arylene; pyrazolyl, isoxazolyl,furyl or thienyl and Y is arylene wherein said pyrazolyl, isoxazolyl,furyl or thienyl is optionally substituted with one or more —C₁-C₄alkyl,C₀₋₄alkyl-C(O)OH, —N(R₁₃)(R₁₄), or halo; or —C(O)N(R₄)(CH₂)₁₋₄—C(O)OR₃and Y is a single bond, —C₁-C₄alkylene or arylene;—C(O)N(R₄)(CH₂)₁₋₄—N(R₁₃)(R₁₄) and Y is a single bond, —C₁-C₄alkylene orarylene; —N(R₄)—C(O)-heteroaryl wherein said heteroaryl is optionallysubstituted with halo and Y is a single bond, —C₁-C₄alkylene or arylene;—N(R₄)—C₁₋₄alkylene-heteroaryl and Y is a single bond, —C₁-C₄alkylene orarylene; —N(R₁₀)—C(O)—[C(R₁₁)(R₁₂)]₁₋₄—N(R₁₃)(R₁₄), and Y is a singlebond, —C₁-C₄alkylene or arylene; —N(R₄)(CH₂)₁₋₄—C(O)OR₄ and Y isarylene; —N(R₄)C(O)C(H)(NH₂)CH₂CH₂—C(O)OR₃ and Y is arylene;—N(R₄)C(O)C(H)(NH₂)CH₂-heteroaryl) and Y is arylene;—N(R₄)C(O)C(H)(CH₃)—NH₂ and Y is arylene; —N(R₄)C(O)CH₂CH₂C(H)(NH₂)—COOHand Y is arylene; —N(R₄)C(O)—C(H)(NH₂)CH₂CH₂—COOH and Y is arylene; or—N(R₄)C(O)-heteroaryl (—N(H)C(O)isoxazolyl) and Y is arylene; iii) R₂ isH, halo or C₁-C₄alkyl; iv) R_(a), R_(a)′, R_(b), R_(b)′, R_(c), R_(d),R_(d)′, R_(e), and R_(e)′ are independently H or C₁-C₄alkyl; V) R₃, R₄,R₅ and R₆ are independently H or C₁-C₄alkyl; vi) R₁₀, R₁₁, R₁₂R₁₃ andR₁₄ are independently H or C₁₋₄alkyl, in free or salt form.
 2. Thecompound according to claim 1, wherein said compound is a compound ofFormula Q-I:

wherein: i) A is S, N(H), CH₂, or O; ii) R₁ is phenyl optionallysubstituted with one or more nitro and/or —N(R₅)(R₆) and Y is a—C₁-C₄alkylene or arylene; phenyl substituted with one or more nitroand/or —N(R₅)(R₆) and Y is a single bond; phenyl substituted with—C(O)OR₃ and Y is a —C₁-C₄alkylene or arylene; heteroaryl wherein saidheteroaryl group is optionally substituted with one or more —C₁-C₄alkyland Y is a single bond, —C₁-C₄alkylene; pyrazolyl, isoxazolyl, furyl orthienyl and Y is arylene wherein said pyrazolyl, isoxazolyl, furyl orthienyl is optionally substituted with one or more —C₁-C₄alkyl,C₀₋₄alkyl-C(O)OH, —N(R₁₃)(R₁₄), or halo; or —C(O)N(R₄)(CH₂)₁₋₄—C(O)OR₃and Y is a single bond, —C₁-C₄alkylene or arylene;—C(O)N(R₄)(CH₂)₁₋₄—N(R₁₃)(R₁₄) and Y is a single bond, —C₁-C₄alkylene orarylene; —N(R₄)—C(O)-heteroaryl wherein said heteroaryl is optionallysubstituted with halo and Y is a single bond, —C₁-C₄alkylene or arylene;—N(R₄)—C₁₋₄alkylene-heteroaryl and Y is a single bond, —C₁-C₄alkylene orarylene; —N(R₁₀)—C(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄) and Y is a single bond,—C₁-C₄alkylene or arylene; —N(R₁₀)—C(O)—C₃₋₁₀heterocycloalkyl and Y is asingle bond, —C₁-C₄alkylene or arylene; —N(R₄)(CH₂)₁₋₄—C(O)OR₄ and Y isarylene; —N(R₄)C(O)C(H)(NH₂)CH₂CH₂—C(O)OR₃ and Y is arylene;—N(R₄)C(O)C(H)(NH₂)CH₂-heteroaryl and Y is arylene;—N(R₄)C(O)C(H)(CH₃)—NH₂ and Y is arylene; —N(R₄)C(O)CH₂CH₂C(H)(NH₂)—COOHand Y is arylene; —N(R₄)C(O)—C(H)(NH₂)CH₂CH₂—COOH and Y is arylene;—N(R₄)C(O)-heteroaryl (—N(H)C(O)isoxazolyl) and Y is arylene; iii) R₂ isH, halo or C₁-C₄alkyl; iv) R_(a), R_(b), R_(b)′, R_(c), R_(d), R_(d)′,R_(e), and R_(e)′ are independently H or C₁-C₄alkyl; v) R₃, R₄, R₅ andR₆ are independently H or C₁-C₄alkyl; vi) R₁₀, R₁₁, R₁₂R₁₃ and R₁₄ areindependently H or C₁₋₄alkyl, in free or salt form.
 3. The compoundaccording to claim 1, wherein said compound is:

wherein R₁ is —N(R₁₀)—C(O)—C(R₁₁)(R₁₂)—N(R₁₃)(R₁₄); R₂ is H or halo;R_(a), R_(a)′, R_(b), R_(b)′, R_(c), R_(d), R_(d)′, R_(e), and R_(e)′are independently H or C₁-C₄alkyl; R₁₀, R₁₁, R₁₂R₁₃ and R₁₄ areindependently H or C₁₋₄alkyl; in free or salt form.
 4. The compoundaccording to claim 1, wherein the compound is a compound selected fromany of the following:

in free or salt form.
 5. The compound according to claim 1, wherein saidcompound is:

in free or salt form.
 6. A pharmaceutical composition comprising acompound according to claim 1, in combination or association with apharmaceutically acceptable diluent or carrier.